Method of Treating Stress Hyperglycemia with Human Antibodies to the Glucagon Receptor

ABSTRACT

The present invention provides antibodies that bind to the human glucagon receptor, designated GCGR and methods of using same. According to certain embodiments of the invention, the antibodies are fully human antibodies that bind to human GCGR. The antibodies of the invention are useful for lowering blood glucose levels and blood ketone levels and are also useful for the treatment of diseases and disorders associated with one or more GCGR biological activities, including the treatment of diabetes, diabetic ketoacidosis, long-term complications associated with diabetes, or other metabolic disorders characterized in part by elevated blood glucose levels, including stress hyperglycemia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 13/301,944, filed on Nov. 22, 2011, which claims the benefitunder 35 U.S.C. §119(e) of U.S. provisional application Ser. No.61/416,409, filed Nov. 23, 2010; U.S. provisional application Ser. No.61/481,958, filed May 3, 2011; and U.S. provisional application Ser. No.61/551,032, filed Oct. 25, 2011. This application also claims thebenefit under 35 U.S.C. §119(e) of U.S. provisional application Ser. No.61/650,966, filed May 23, 2012; and U.S. provisional application Ser.No. 61/787,748, filed Mar. 15, 2013. The disclosures of all theforegoing are herein specifically incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The present invention is related to methods of using a glucagon receptorantagonist for treating stress hyperglycemia in critically ill andnon-critically ill patients suffering from such condition.

STATEMENT OF RELATED ART

Glucagon is a 29 amino acid hormone produced by the alpha cells ofpancreatic islets. Glucagon maintains normal levels of glucose inanimals, including humans, by counter-balancing the effects of insulin.It is an imbalance of glucagon and insulin that may play an importantrole in several diseases, such as diabetes mellitus and diabeticketoacidosis. In particular, studies have shown that higher basalglucagon levels and lack of suppression of postprandial glucagonsecretion contribute to diabetic conditions in humans (Muller et al., NEng J Med 283: 109-115 (1970)).

It is believed that glucagon's effects on elevating blood glucose levelsare mediated in part by the activation of certain cellular pathwaysfollowing the binding of glucagon (GCG) to its receptor (designatedGCGR). GCGR is a member of the secretin subfamily (family B) ofG-protein-coupled receptors and is predominantly expressed in the liver.The binding of glucagon to its receptor triggers a G-protein signaltransduction cascade, activating intracellular cyclic AMP and leading toan increase in glucose output through de novo synthesis(gluconeogenesis) and glycogen breakdown (glycogenolysis) (Wakelam etal., Nature, (1986) 323:68-71; Unson et al., Peptides, (1989),10:1171-1177; and Pittner and Fain, Biochem. J. (1991), 277:371-378).

The rat glucagon receptor was first isolated and purified by Jelinek etal (Jelinek, L. J. et al. (1993) Science 259(5101): 1614-1616).Subsequently, the rat sequence was used to identify and clone the 477amino acid human glucagon receptor sequence (Lok, S. et al. (1994) Gene140:203-209; MacNeil, D. J. et al. (1994) Biochem. and Biophys. Res.Comm). U.S. Pat. No. 5,776,725 discloses an isolated nucleic acidsequence encoding a human or rat glucagon receptor.

Targeting glucagon production or function with a glucagon receptorantagonist, such as an anti-GCGR antibody, may be one method ofcontrolling and lowering blood glucose, and as such, may prove usefulfor treating diseases such as diabetes mellitus or diabeticketoacidosis. Furthermore, by lowering glucose levels, it may bepossible to prevent or ameliorate certain of the long-term complicationsassociated with elevated glucose levels in diabetic patients.

Early studies demonstrated that polyclonal antibodies to the ratglucagon receptor were able to block glucagon binding (Unson, C. G.(1996) PNAS 93(1):310-315). Monoclonal antibodies to the human glucagonreceptor were described by Buggy et al. (Buggy, J. J. et al. (1995) J.Biol. Chem. 270(13): 7474; Buggy, J. J. et al. (1996) Horm Metab Res.28(5):215-9). The antibody described by Buggy et al. competed withglucagon for the hormone binding site of the receptor and recognizedboth the human and rat glucagon receptors, but not the mouse receptor.Wright et al. disclose a monoclonal antibody raised in a mouse againstthe human glucagon receptor and conducted detailed protein structuredetermination of the monoclonal antibody to the receptor (Wright, L. M.(2000) Acta Crystallographica Section D. 56(5): 573-580). Otherantibodies to the glucagon receptor are described in U.S. Pat. Nos.5,770,445 and 7,947,809; European patent application EP2074149A2; EPpatent EP0658200B1; US patent publications 2009/0041784; 2009/0252727;and 2011/0223160; and PCT publication WO2008/036341.

Stress hyperglycemia (also referred to as stress-induced hyperglycemia),which is defined as a transient increase in blood glucose (>140 mg/dL),is a condition that is temporally linked to the stress of an acuteinjury or illness. This common medical condition occurs frequently inthe diabetic and non-diabetic hospitalized patient and requires prompttherapeutic intervention (Kitabchi A E, Umpierrez G E, Miles J M, FisherJ N, Diabetes Care, (2009), July; 32(7):1335-43; Baker E H, Janaway C H,Philips B J, Brennan A L, Baines D L, Wood D M, et al., Thorax, (2006),April; 61(4):284-9). Serious medical and surgical conditions associatedwith stress hyperglycemia include myocardial infarction, burns, andcardiopulmonary bypass surgery (Rocha D M, Santeusanio F, Faloona G R,Unger R H, N Engl J. Med. (1973), Apr. 5; 288(14):700-3). Stresshyperglycemia also occurs in non-critically ill patients, for example,in those admitted for exacerbation of chronic obstructive pulmonarydisease (Oshima C, Kaneko T, Tsuruta R, Oda Y, Miyauchi T, Fujita M, etal., Resuscitation, (2010), February 81(2):187-92). In hospitalizedpatients, stress hyperglycemia is associated with substantial increasesin infections, dialysis, blood transfusions, polyneuropathy, and othermedical complications including death (up to 18-fold).

Stress hyperglycemia has increasingly been the focus of clinicalinvestigations that demonstrate reductions in the aforementioned medicalcomplications with intensive insulin therapy. However, intensive insulintherapy is associated with a substantial risk of hypoglycemia, whichgreatly reduces the medical benefits of therapy. As a result, currentpractice guidelines have been revised to target higher blood glucoselevels in order to lower the risk of hypoglycemia, but this approach maylimit the benefit of therapy (Kitabchi A E, Umpierrez G E, Miles J M,Fisher J N, Diabetes Care, (2009), July 32(7):1335-43; Baker E H,Janaway C H, Philips B J, Brennan A L, Baines D L, Wood D M, et al.,Thorax, (2006), April 61(4):284-9).

Studies in patients admitted for conditions associated with stresshyperglycemia have demonstrated sustained glucagon elevations up to10-fold above the normal levels (Rocha D M, Santeusanio F, Faloona G R,Unger R H, N Engl J. Med. (1973), Apr. 5; 288(14):700-3); Oshima C,Kaneko T, Tsuruta R, Oda Y, Miyauchi T, Fujita M, et al., Resuscitation,(2010), February 81(2):187-92). In these settings, elevated glucagon maycontribute to elevated blood glucose levels and may hamper achievingtarget glycemic control.

Currently, there are no therapies specifically approved by regulatoryagencies to prevent the onset of, or to treat stress hyperglycemia.Stress hyperglycemia in patients who are critically ill represents aserious unmet medical need for several reasons. In particular, thesepatients have a high risk for imminent death since existing therapies,i.e., intensive insulin, may paradoxically increase the risk for deathby causing severe hypoglycemia. Moreover, the etiology of stresshyperglycemia is multifactorial and insulin therapy alone may beinsufficient for optimal control. Furthermore, while non-critically illpatients may have a low risk for death, they may still benefit fromimproved metabolic control and would thus experience lower morbidity.

Therefore, there is a need for new therapies, which may be used alone,or as adjunct therapy with other glucose lowering agents for treatingstress hyperglycemia in order to reduce the risk of hypoglycemicepisodes and to reduce the morbidity and mortality associated with thiscondition.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention provides fully human monoclonalantibodies (mAbs) and antigen-binding fragments thereof that bind to thehuman glucagon receptor (hGCGR) and inhibit or block its activity, forexample, block the binding of glucagon to its receptor, thereby blockingthe elevation of blood glucose levels. The antibodies or antigen bindingfragments thereof may be useful for lowering blood glucose levels in asubject that suffers from a disease characterized by increased bloodglucose levels, such as diabetes mellitus. The antibodies may also beused to treat a wide range of conditions and disorders in which blockingthe interaction of glucagon with the glucagon receptor is desired,thereby having a beneficial effect. The antibodies may ultimately beused to prevent the long-term complications associated with elevatedblood glucose levels in diabetic patients, or to ameliorate at least onesymptom associated with elevated blood glucose levels in diabeticpatients. The antibodies of the present invention may also be used toaddress the unmet need for new therapies to prevent the onset of stresshyperglycemia, or to treat stress hyperglycemia, when used alone tolower blood glucose levels, or as adjunct therapy with at least oneother glucose lowering agent, for example, insulin. Furthermore, theantibodies of the present invention may prove beneficial in loweringblood glucose levels without the risk of inducing hypoglycemia inpatients suffering from stress hyperglycemia. In addition, the use ofthe antibodies of the invention may prove beneficial in lowering therisk of infections, organ failure, or morbidity and/or mortality inpatients suffering from stress hyperglycemia.

The antibodies of the invention can be full-length (for example, an IgG1or IgG4 antibody) or may comprise only an antigen-binding portion (forexample, a Fab, F(ab′)₂ or scFv fragment), and may be modified to affectfunctionality, e.g., to eliminate residual effector functions (Reddy etal., 2000, J. Immunol. 164:1925-1933).

In one embodiment, the invention provides an isolated human antibody orantigen-binding fragment thereof that specifically binds human glucagonreceptor (hGCGR), wherein the antibody binds an ectodomain and/or anextracellular (EC) loop of human GCGR, wherein the ectodomain is theN-terminal domain of GCGR and wherein the EC loop is one or more of EC1,EC2 and EC3.

In one embodiment, the invention provides an antibody or fragmentthereof, which binds the N-terminal domain comprising amino acidresidues ranging from about amino acid residue number 27 to about aminoacid residue 144 of SEQ ID NO: 153, or binds an EC loop of hGCGR,wherein the EC loop is one or more of EC1, EC2, and EC3, wherein EC1comprises amino acid residues ranging from about amino acid residue 194to about amino acid residue 226 of SEQ ID NO: 153; EC2 comprises aminoacid residues ranging from about amino acid residue 285 to about aminoacid residue 305 of SEQ ID NO: 153; and EC3 comprises amino acidresidues ranging from about amino acid residue 369 to about amino acidresidue 384 of SEQ ID NO: 153.

In one embodiment, the human antibody or antigen-binding fragment of ahuman antibody that binds hGCGR, comprises a heavy chain variable region(HCVR) having an amino acid sequence selected from the group consistingof SEQ ID NO: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130 and 146,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity. In certainembodiments, the antibody or antigen-binding fragment of an antibodythat binds hGCGR comprises a HCVR having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 34, 70, 86, 110 and 126, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity.

In one embodiment, the human antibody or antigen-binding fragment of ahuman antibody that binds hGCGR comprises a light chain variable region(LCVR) having an amino acid sequence selected from the group consistingof SEQ ID NO: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138 and148, or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity. In certainembodiments, the antibody or antigen-binding fragment of an antibodythat binds hGCGR comprises a LCVR having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 42, 78, 88, 118 and 128, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity.

In certain embodiments, the human antibody or fragment thereof thatbinds hGCGR comprises a HCVR/LCVR amino acid sequence pair selected fromthe group consisting of SEQ ID NO: 2/10, 18/26, 34/42, 50/58, 66/68,70/78, 86/88, 90/98, 106/108, 110/118, 126/128, 130/138, and 146/148. Incertain embodiments, the HCVR/LCVR amino acid sequence pair is selectedfrom the group consisting of SEQ ID NO: 34/42, 70/78, 86/88, 110/118 and126/128.

In a related embodiment, the invention includes an antibody orantigen-binding fragment of an antibody which specifically binds hGCGR,wherein the antibody or fragment thereof comprises the heavy and lightchain CDR domains contained within heavy and light chain sequence pairsselected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42,50/58, 66/68, 70/78, 86/88, 90/98, 106/108, 110/118, 126/128, 130/138and 146/148. Methods and techniques for identifying CDRs within HCVR andLCVR amino acid sequences are well known in the art and can be used toidentify CDRs within the specified HCVR and/or LCVR amino acid sequencesdisclosed herein. Exemplary conventions that can be used to identify theboundaries of CDRs include, e.g., the Kabat definition, the Chothiadefinition, and the AbM definition. In general terms, the Kabatdefinition is based on sequence variability, the Chothia definition isbased on the location of the structural loop regions, and the AbMdefinition is a compromise between the Kabat and Chothia approaches.See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,”National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al.,J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad.Sci. USA 86:9268-9272 (1989). Public databases are also available foridentifying CDR sequences within an antibody.

In certain embodiments, the present invention provides an isolated humanantibody or an antigen-binding fragment thereof that binds specificallyto hGCGR, wherein the antibody comprises a HCVR comprising the threeheavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within the HCVRsequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50,66, 70, 86, 90, 106, 110, 126, 130 and 146; and a LCVR comprising thethree light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within theLCVR sequences selected from the group consisting of SEQ ID NO: 10, 26,42, 58, 68, 78, 88, 98, 108, 118, 128, 138 and 148.

In one embodiment, the present invention provides an isolated humanantibody or antigen-binding fragment of a human antibody that bindshGCGR, comprising a HCDR3 domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 8, 24, 40, 56, 76, 96, 116 and136, or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; and a LCDR3domain having an amino acid sequence selected from the group consistingof SEQ ID NO: 16, 32, 48, 64, 84, 104, 124 and 144, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

In one embodiment, the invention provides an antibody or fragmentthereof that further comprises a HCDR1 domain having an amino acidsequence selected from the group consisting of SEQ ID NO: 4, 20, 36, 52,72, 92, 112 and 132, or a substantially similar sequence thereof havingat least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; a HCDR2 domain having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 6, 22, 38, 54, 74, 94, 114 and 134, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; a LCDR1 domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 12, 28, 44, 60, 80, 100, 120 and 140, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; and a LCDR2 domain having an amino acidsequence selected from the group consisting of SEQ ID NO: 14, 30, 46,62, 82, 102, 122 and 142, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

In one embodiment, the antibody or antigen-binding fragment of anantibody comprises:

(a) a HCDR3 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 8, 24, 40, 56, 76, 96, 116 and 136; and

(b) a LCDR3 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 16, 32, 48, 64, 84, 104, 124 and 144.

In one embodiment, the antibody or antigen-binding fragment of theantibody further comprises:

(c) a HCDR1 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, 20, 36, 52, 72, 92, 112 and 132;

(d) a HCDR2 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 6, 22, 38, 54, 74, 94, 114 and 134;

(e) a LCDR1 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 12, 28, 44, 60, 80, 100, 120 and 140; and

(f) a LCDR2 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 14, 30, 46, 62, 82, 102, 122 and 142.

In one embodiment, the antibody or antigen-binding fragment thereofcomprises a HCVR comprising a HCDR1 domain having an amino acid sequenceselected from one of SEQ ID NO: 4, 20, 36, 52, 72, 92, 112 and 132; aHCDR2 domain having an amino acid sequence selected from one of SEQ IDNO: 6, 22, 38, 54, 74, 94, 114 and 134; a HCDR3 domain having an aminoacid sequence selected from one of SEQ ID NOs: 8, 24, 40, 56, 76, 96,116 and 136; and a LCVR comprising a LCDR1 domain having an amino acidsequence selected from one of SEQ ID NO: 12, 28, 44, 60, 80, 100, 120and 140; a LCDR2 domain having an amino acid sequence selected from oneof SEQ ID NO: 14, 30, 46, 62, 82, 102, 122 and 142; and a LCDR3 domainhaving an amino acid sequence selected from one of SEQ ID NO: 16, 32,48, 64, 84, 104, 124 and 144.

In certain embodiments, the human antibody or antigen-binding fragmentof a human antibody that binds to human GCGR comprises a HCDR3/LCDR3amino acid sequence pair selected from the group consisting of SEQ IDNO: 8/16, 24/32, 40/48, 56/64, 76/84, 96/104, 116/124 and 136/144.Non-limiting examples of anti-GCGR antibodies having these HCDR3/LCDR3pairs are the antibodies designated H4H1345N, H4H1617N, H4H1765N,H4H1321B and H4H1321P, H4H1327B and H4H1327P, H4H1328B and H4H1328P,H4H1331B and H4H1331P, H4H1339B and H4H1339P, respectively.

In one embodiment, the human antibody or antigen binding fragmentthereof comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQID NO: 4, 6 and 8, respectively and LCDR1, LCDR2 and LCDR3 amino acidsequences of SEQ ID NO: 12, 14 and 16, respectively.

In one embodiment, the human antibody or antigen binding fragmentthereof comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQID NO: 20, 22 and 24, respectively and LCDR1, LCDR2 and LCDR3 amino acidsequences of SEQ ID NO: 28, 30 and 32, respectively.

In one embodiment, the human antibody or antigen binding fragmentthereof comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQID NO: 36, 38 and 40, respectively and LCDR1, LCDR2 and LCDR3 amino acidsequences of SEQ ID NO: 44, 46 and 48, respectively.

In one embodiment, the human antibody or antigen binding fragmentthereof comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQID NO: 52, 54 and 56, respectively and LCDR1, LCDR2 and LCDR3 amino acidsequences of SEQ ID NO: 60, 62 and 64, respectively.

In one embodiment, the human antibody or antigen binding fragmentthereof comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQID NO: 72, 74 and 76, respectively and LCDR1, LCDR2 and LCDR3 amino acidsequences of SEQ ID NO: 80, 82 and 84, respectively.

In one embodiment, the human antibody or antigen binding fragmentthereof comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQID NO: 92, 94 and 96, respectively and LCDR1, LCDR2 and LCDR3 amino acidsequences of SEQ ID NO: 100, 102 and 104, respectively.

In one embodiment, the human antibody or antigen binding fragmentthereof comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQID NO: 112, 114 and 116, respectively and LCDR1, LCDR2 and LCDR3 aminoacid sequences of SEQ ID NO: 120, 122 and 124, respectively.

In one embodiment, the human antibody or antigen binding fragmentthereof comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQID NO: 132, 134 and 136, respectively and LCDR1, LCDR2 and LCDR3 aminoacid sequences of SEQ ID NO: 140, 142 and 144, respectively.

In one embodiment, the anti-hGCGR antibody or antigen binding fragmentthereof comprises a HCDR1 sequence comprising the formulaX¹—X²—X³—X⁴—X⁵—X⁶—X⁷—X⁸ (SEQ ID NO: 202), wherein X¹ is Gly, X² is Phe,X³ is Thr, X⁴ is Phe or Ser, X⁵ is Ser, X⁶ is Ser or Asn, X⁷ is Tyr orPhe, and X⁸ is Asp, Leu, or Gly; a HCDR2 sequence comprising the formulaX¹—X²—X³—X⁴—X⁵—X⁶—X⁷—X⁸ (SEQ ID NO: 203), wherein X¹ is Ile, X² is Ser,Gln, Asp, or Trp, X³ is Ser, Glu, Thr, or Phe, X⁴ is Asp or Ala, X⁵ isGly or Glu, X⁶ is Arg, Ile, or absent, X⁷ is Asp or Glu, and X⁸ is Lysor Thr; a HCDR3 sequence comprising the formulaX¹—X²—X³—X⁴—X⁵—X⁶—X⁷—X⁸—X⁹—X¹⁰—X¹¹—X¹²—X¹³—X¹⁴—X¹⁵—X¹⁶—X¹⁷—X¹⁸—X¹⁹—X²⁰—X²¹(SEQ ID NO: 204), wherein X¹ is Ala or Thr, X² is Lys or Arg, X³ is Glu,X⁴ is Met, Pro, Gly, or Asp, X⁵ is Val, Ser, Lys, Arg, or absent, X⁶ isTyr, His, Asn, or absent, X⁷ is Tyr, X⁸ is Asp or Glu, X⁹ is Ile, X¹⁰ isLeu, X¹¹ is Thr, X¹² is Gly, X¹³ is Tyr, Asp, or His, X¹⁴ is His, Asp,Tyr, or absent, X¹⁵ is Asn, Tyr, His, or absent, X¹⁶ is Tyr, X¹⁷ is Tyror His, X¹⁸ is Gly or Ala, X¹⁹ is Met, X²⁰ is Asp and X²¹ is Val or Ile;a LCDR1 sequence comprising the formula X¹—X²—X³—X⁴—X⁵—X⁶ (SEQ ID NO:205), wherein X¹ is Gln, X² is Gly or Ala, X³ is Ile, X⁴ is Asn or Arg,X⁵ is Asn, and X⁶ is Tyr or Asp; a LCDR2 sequence comprising the formulaX¹—X²—X³ (SEQ ID NO: 206), wherein X¹ is Thr or Ala, X² is Ala or Thr,and X³ is Ser or Phe; and a LCDR3 sequence comprising the formulaX¹—X²—X³—X⁴—X⁵—X⁶—X⁷—X⁸—X⁹ (SEQ ID NO: 207), wherein X¹ is Gln or Leu,X² is Gln, X³ is Tyr, His, or Asp, X⁴ is Asn or Tyr, X⁵ is Thr or Ser,X⁶ is Tyr, Asn, or His, X⁷ is Pro, X⁸ is Leu, Phe, Arg, or absent and X⁹is Thr.

In one embodiment, the antibody or antigen-binding fragment binds human,monkey, mouse and rat GCGR.

In one embodiment, the antibody or antigen-binding fragment binds human,monkey and mouse GCGR, but does not bind rat GCGR.

In one embodiment, the antibody or antigen-binding fragment binds human,monkey and rat GCGR, but does not bind mouse GCGR.

In one embodiment, the antibody or antigen-binding fragment binds humanand monkey GCGR, but does not bind rat or mouse GCGR.

In one embodiment, the antibody or antigen-binding fragment binds humanGCGR, but does not bind monkey, mouse or rat GCGR.

In one embodiment, the invention provides a fully human monoclonalantibody or antigen-binding fragment thereof that neutralizes hGCGRactivity, wherein the antibody or fragment thereof exhibits one or moreof the following characteristics: (i) comprises a HCVR having an aminoacid sequence selected from the group consisting of SEQ ID NO: 2, 18,34, 50, 66, 70, 86, 90, 106, 110, 126, 130 and 146; (ii) comprises aLCVR having an amino acid sequence selected from the group consisting ofSEQ ID NO: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138 and 148;(iii) comprises a HCDR3 domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 8, 24, 40, 56, 76, 96, 116 and136, or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; and a LCDR3domain having an amino acid sequence selected from the group consistingof SEQ ID NO: 16, 32, 48, 64, 84, 104, 124 and 144, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; (iv) comprises a HCDR1 domain havingan amino acid sequence selected from the group consisting of SEQ ID NO:4, 20, 36, 52, 72, 92, 112 and 132, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity; a HCDR2 domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 6, 22, 38, 54, 74, 94, 114 and134, or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; a LCDR1domain having an amino acid sequence selected from the group consistingof SEQ ID NO: 12, 28, 44, 60, 80, 100, 120 and 140, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; and a LCDR2 domain having an aminoacid sequence selected from the group consisting of SEQ ID NO: 14, 30,46, 62, 82, 102, 122 and 142, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity; (v) binds any one or more of human, monkey, mouse orrat GCGR; (vi) may or may not block GCGR activity in at least onespecies other than human; (v) demonstrates a K_(D) ranging from about10⁻⁸ to about 10⁻¹²; (vi) lowers blood glucose levels by at least about25% to about 75% in a mammal experiencing elevated blood glucose levels;(vii) may or may not lower triglyceride levels to levels observed in anormal mammal; or (viii) demonstrates no adverse effect on blood levelsof LDL, HDL, or total cholesterol in a mammal.

In another related embodiment, the invention provides an antibody orantigen-binding fragment thereof that competes for specific binding tohGCGR with an antibody or antigen-binding fragment comprising thecomplementarity determining regions (CDRs) of a heavy chain variableregion (HCVR), wherein the HCVR has an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106,110, 126, 130 and 146; and the CDRs of a light chain variable region(LCVR), wherein the LCVR has an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108,118, 128, 138 and 148.

In one embodiment, the invention provides an antibody or antigen-bindingfragment thereof that competes for specific binding to hGCGR with anantibody or antigen-binding fragment comprising heavy and light chainCDR domains contained within heavy and light chain sequence pairsselected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42,50/58, 66/68, 70/78, 86/88, 90/98, 106/108, 110/118, 126/128, 130/138and 146/148.

In another related embodiment the invention provides an antibody orantigen-binding fragment thereof that binds the same epitope on hGCGR asan antibody or antigen-binding fragment comprising the complementaritydetermining regions (CDRs) of a heavy chain variable region (HCVR),wherein the HCVR has an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126,130 and 146; and the CDRs of a light chain variable region (LCVR),wherein the LCVR has an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128,138 and 148.

In one embodiment, the invention provides an antibody or antigen-bindingfragment thereof that binds the same epitope on hGCGR that is recognizedby an antibody comprising heavy and light chain sequence pairs selectedfrom the group consisting of SEQ ID NO: 2/10, 18/26, 34/42, 50/58,66/68, 70/78, 86/88, 90/98, 106/108, 110/118, 126/128, 130/138 and146/148.

In one embodiment, the invention provides for an anti-hGCGR antibodyhaving one or more of the following characteristics:

-   -   a) capable of reducing blood glucose levels by about 25% to        about 75% for a period of at least 7 days, when administered at        a dose ranging from about 1 mg/kg to about 30 mg/kg;    -   b) capable of resulting in at least a 10% reduction in body        weight when administered to a mammal in need of such therapy;    -   c) capable of reducing blood ketone levels by about 25% to 75%        when administered at a dose ranging from about 1 mg/kg to about        30 mg/kg; or    -   d) capable of reducing blood glucose levels by about 20% to        about 40% without causing a significant elevation in blood        lipids or cholesterol when administered with an antibody        specific for proprotein convertase subtilisin/kexin type        (PCSK)-9, and sustaining lowered blood glucose levels for at        least 7 days post treatment.

In a second aspect, the invention provides nucleic acid moleculesencoding anti-hGCGR antibodies or fragments thereof. Recombinantexpression vectors carrying the nucleic acids of the invention, and hostcells into which such vectors have been introduced, are also encompassedby the invention, as are methods of producing the antibodies byculturing the host cells under conditions permitting production of theantibodies, and recovering the antibodies produced.

In one embodiment, the invention provides an antibody or fragmentthereof comprising a HCVR encoded by a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 1, 17, 33, 49, 65, 69, 85, 89,105, 109, 125, 129 and 145, or a substantially identical sequence havingat least 90%, at least 95%, at least 98%, or at least 99% homologythereof. In one embodiment, the HCVR is encoded by a nucleic acidsequence selected from the group consisting of SEQ ID NO: 33, 69, 85,109 and 125.

In one embodiment, the antibody or fragment thereof further comprises aLCVR encoded by a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 9, 25, 41, 57, 67, 77, 87, 97, 107, 117, 127,137 and 147, or a substantially identical sequence having at least 90%,at least 95%, at least 98%, or at least 99% homology thereof. In oneembodiment, the LCVR is encoded by a nucleic acid sequence selected fromthe group consisting of SEQ ID NO: 41, 77, 87, 117 and 127.

In one embodiment, the invention also provides an antibody orantigen-binding fragment of an antibody comprising a HCDR3 domainencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO: 7, 23, 39, 55, 75, 95, 115 and 135, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; and a LCDR3 domain encoded by anucleotide sequence selected from the group consisting of SEQ ID NO: 15,31, 47, 63, 83, 103, 123 and 143, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity.

In one embodiment, the invention provides an antibody or fragmentthereof further comprising a HCDR1 domain encoded by a nucleotidesequence selected from the group consisting of SEQ ID NO: 3, 19, 35, 51,71, 91, 111 and 131, or a substantially similar sequence thereof havingat least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; a HCDR2 domain encoded by a nucleotide sequence selected fromthe group consisting of SEQ ID NO: 5, 21, 37, 53, 73, 93, 113 and 133,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; a LCDR1domain encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 11, 27, 43, 59, 79, 99, 119 and 139, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; and a LCDR2 domainencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO: 13, 29, 45, 61, 81, 101, 121 and 141, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

In a third aspect, the invention features a human anti-hGCGR antibody orantigen-binding fragment of an antibody comprising a HCVR encoded bynucleotide sequence segments derived from V_(H), D_(H) and J_(H)germline sequences, and a LCVR encoded by nucleotide sequence segmentsderived from V_(K) and J_(K) germline sequences, with combinations asshown in Table 2.

The invention encompasses anti-hGCGR antibodies having a modifiedglycosylation pattern. In some applications, modification to removeundesirable glycosylation sites may be useful, or e.g., removal of afucose moiety to increase antibody dependent cellular cytotoxicity(ADCC) function (see Shield et al. (2002) JBC 277:26733). In otherapplications, modification of galactosylation can be made in order tomodify complement dependent cytotoxicity (CDC).

In a fourth aspect, the invention features a pharmaceutical compositioncomprising a recombinant human antibody or fragment thereof, whichspecifically binds hGCGR, and a pharmaceutically acceptable carrier ordiluent.

In one embodiment, the invention features a composition, which is acombination of an antibody or antigen-binding fragment of an antibody ofthe invention, and a second therapeutic agent. The second therapeuticagent may be any agent that is advantageously combined with the antibodyor fragment thereof of the invention.

In one embodiment, the second therapeutic agent may be an agent capableof lowering blood glucose or reducing at least one symptom in a patientsuffering from a disease or condition characterized by high bloodglucose levels, such as diabetes mellitus.

In certain embodiments, the second therapeutic agent may be an agentthat helps to counteract or reduce any possible side effect(s)associated with the antibody or antigen-binding fragment of an antibodyof the invention, if such side effect(s) should occur. For example, inthe event that any of the anti-hGCGR antibodies increases lipid orcholesterol levels, it may be beneficial to administer a second agentthat is effective to lower lipid or cholesterol levels.

The second therapeutic agent may be a small molecule drug, aprotein/polypeptide, an antibody, a nucleic acid molecule, such as ananti-sense molecule, or a siRNA. The second therapeutic agent may besynthetic or naturally derived.

In one embodiment, the second therapeutic agent may be a glucagonantagonist, or a second glucagon receptor antagonist, such as anotherantibody to the glucagon receptor, which is different than theantibodies described herein. It will also be appreciated that theantibodies and pharmaceutically acceptable compositions of the presentinvention can be employed in combination therapies, that is, theantibodies and pharmaceutically acceptable compositions can beadministered concurrently with, prior to, or subsequent to, one or moreother desired therapeutics or medical procedures. The particularcombination of therapies (therapeutics or procedures) to employ in acombination regimen will take into account compatibility of the desiredtherapeutics and/or procedures and the desired therapeutic effect to beachieved. It will also be appreciated that the therapies employed mayachieve a desired effect for the same disorder (for example, an antibodymay be administered concurrently with another agent used to treat thesame disorder), or they may achieve different effects (e.g., control ofany adverse effects). As used herein, additional therapeutic agents thatare normally administered to treat or prevent a particular disease, orcondition, are appropriate for the disease, or condition, being treated.

In one embodiment, the anti-hGCGR antibodies of the invention may beused in combination with one or more of the following type 2 diabetestreatments currently available. These include biguanide (metformin),sulfonylureas (such as glyburide, glipizide), peroxisomeproliferator-activated receptor (PPAR) gamma agonists (pioglitazone,rosiglitazone); and alpha glucosidase inhibitors (acarbose, voglibose).Additional treatments include injectable treatments such as EXENATIDE®(glucagon-like peptide 1), and SYMLIN® (pramlintide).

In certain embodiments, the composition may include a second agentselected from the group consisting of non-sulfonylurea secretagogues,insulin, insulin analogs, exendin-4 polypeptides, beta 3 adrenoceptoragonists, PPAR agonists, dipeptidyl peptidase IV inhibitors, statins andstatin-containing combinations, inhibitors of cholesterol uptake and/orbile acid re-absorption, LDL-cholesterol antagonists, cholesteryl estertransfer protein antagonists, endothelin receptor antagonists, growthhormone antagonists, insulin sensitizers, amylin mimetics or agonists,cannabinoid receptor antagonists, glucagon-like peptide-1 agonists,melanocortins, melanin-concentrating hormone receptor agonists, SNRIs, afibroblast growth factor 21 (FGF21) mimetic (See, for example,US20110002845 and US20080261236), a fibroblast growth factor receptor 1c(FGFR1c) agonist (See, for example, US20110150901), an inhibitor ofadvanced glycation endproduct formation, such as, but not limited to,aminoguanidine, and protein tyrosine phosphatase inhibitors.

In certain embodiments, the composition may include a second agent tohelp lower lipid or cholesterol levels and may include an agent such asa 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor(for example, a statin such as atorvastatin, (LIPITOR®), fluvastatin(LESCOL®), lovastatin (MEVACOR®), pitavastatin (LIVALO®), pravastatin(PRAVACHOL®), rosuvastatin (CRESTOR®) and simvastatin (ZOCOR®) and thelike.

In certain embodiments, it may be beneficial to administer theantibodies of the invention in combination with any one or more of thefollowing: (1) niacin, which increases lipoprotein catabolism; (2)fibrates or amphipathic carboxylic acids, which reduce low-densitylipoprotein (LDL) level, improve high-density lipoprotein (HDL) andtriglycerides (TG) levels, and reduce the number of non-fatal heartattacks; and (3) activators of the LXR transcription factor that plays arole in cholesterol elimination such as 22-hydroxycholesterol, or fixedcombinations such as VYTORIN®) (ezetimibe plus simvastatin); a statinwith a bile resin (e.g., cholestyramine, colestipol, colesevelam), afixed combination of niacin plus a statin (e.g., niacin withlovastatin); or with other lipid lowering agents such as omega-3-fattyacid ethyl esters (for example, omacor). Furthermore, the secondtherapeutic agent can be one or more other inhibitors of glucagon orhGCGR, as well as inhibitors of other molecules, such asangiopoietin-like protein 3 (ANGPTL3), angiopoietin-like protein 4(ANGPTL4), angiopoietin-like protein 5 (ANGPTL5), angiopoietin-likeprotein 6 (ANGPTL6), which are involved in lipid metabolism, inparticular, cholesterol and/or triglyceride homeostasis. Inhibitors ofthese molecules include small molecules and antibodies that specificallybind to these molecules and block their activity.

In certain embodiments, it may be beneficial to administer the anti-GCGRantibodies of the invention in combination with a nucleic acid thatinhibits the activity of hPCSK9, such as an antisense molecule, a doublestranded RNA, or a siRNA molecule. Exemplary nucleic acid molecules thatinhibit the activity of PCSK9 are described in US2011/0065644,US2011/0039914, US2008/0015162 and US2007/0173473.

In certain embodiments, it may be beneficial to administer theanti-hGCGR antibodies of the invention in combination with an antibodythat specifically binds to and inhibits the activity of hPCSK9, whereinsuch antibody acts to lower lipid or cholesterol levels. Exemplaryanti-hPCSK9 antibodies are described in US2010/0166768. The isolatedantibody that specifically binds to human PCSK9, or an antigen-bindingfragment thereof, may be administered at a dose ranging from about 0.01mg/kg to about 30 mg/kg. It may be administered as a single dose or asmultiple doses. The anti-hPCSK9 antibody may be administeredconcurrently with the anti-GCGR antibody, or it may be administeredprior to, or after the anti-GCGR antibody.

In one embodiment, the second therapeutic agent to be used incombination with an antibody of the invention comprises an isolatedantibody that specifically binds to human PCSK9, or an antigen-bindingfragment thereof, wherein the anti-h PCSK9 antibody comprises the threeheavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within any one ofthe HCVR sequences selected from the group consisting of SEQ ID NOs: 173and 177; and the three light chain CDRs (LCDR1, LCDR2 and LCDR3)contained within any one of the LCVR sequences selected from the groupconsisting of SEQ ID NOs: 175 and 185.

In one embodiment, the isolated antibody that specifically binds tohuman PCSK9, or antigen-binding fragment thereof, comprises a heavychain variable region (HCVR) selected from the group consisting of SEQID NO: 173 and 177.

In one embodiment, the isolated antibody that specifically binds tohuman PCSK9, or antigen-binding fragment thereof, comprises a lightchain variable region (LCVR) selected from the group consisting of SEQID NO: 175 and 185.

In one embodiment, the isolated antibody that specifically binds tohuman PCSK9, or antigen-binding fragment thereof, comprises a HCVRhaving an amino acid sequence selected from the group consisting of SEQID NO: 173 and 177 and a LCVR having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 175 and 185.

In one embodiment, the isolated antibody that specifically binds tohuman PCSK9, or antigen-binding fragment thereof, comprises a heavychain variable region (HCVR) and a light chain variable region (LCVR),wherein the HCVR/LCVR sequence pairs are selected from the groupconsisting of SEQ ID NO: 173/175; and SEQ ID NO: 177/185.

In one embodiment, the isolated antibody that specifically binds tohuman PCSK9, or antigen-binding fragment thereof, comprises: a HCDR1comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 161 and 179; a HCDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 163 and 181; a HCDR3comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 165 and 183; a LCDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 167 and 187; a LCDR2comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 169 and 189 and a LCDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 171 and 191.

In certain embodiments, the hPCSK9 antibodies to be used in combinationwith the anti-GCGR antibodies of the invention are encoded by nucleicacid molecules as described herein. For example, in one embodiment, theinvention provides an anti-h PCSK9 antibody or fragment thereofcomprising a HCVR encoded by a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 172 and 176, or a substantially identicalsequence having at least 90%, at least 95%, at least 98%, or at least99% homology thereof, and a LCVR encoded by a nucleic acid sequenceselected from the group consisting of SEQ ID NO: 174 and 184, or asubstantially identical sequence having at least 90%, at least 95%, atleast 98%, or at least 99% homology thereof.

In one embodiment, the invention provides an anti-hPCSK9 antibody to beused in combination with the anti-GCGR antibodies of the invention,wherein the anti-PCSK9 antibody or fragment thereof comprises a HCDR1domain encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 160 and 178, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; a HCDR2 domain encoded by a nucleotidesequence selected from the group consisting of SEQ ID NO: 162 and 180,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; a HCDR3domain encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 164 and 182, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; a LCDR1 domain encoded by a nucleotidesequence selected from the group consisting of SEQ ID NO: 166 and 186,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; a LCDR2domain encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 168 and 188, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; and a LCDR3 domain encoded by a nucleotidesequence selected from the group consisting of SEQ ID NO: 170 and 190,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity.

When multiple therapeutics are co-administered, dosages may be adjustedaccordingly, as is recognized in the pertinent art.

In a fifth aspect, the invention features methods for inhibiting hGCGRactivity using the anti-hGCGR antibody or antigen-binding portion of theantibody of the invention, wherein the therapeutic methods compriseadministering a therapeutically effective amount of a pharmaceuticalcomposition comprising the antibody or antigen-binding fragment thereof.The antibodies of the invention may be used to treat any condition ordisorder, which is improved, ameliorated, inhibited or prevented byremoval, inhibition or reduction of hGCGR activity. It is envisionedthat the antibodies of the invention may be used alone, or as adjuncttherapy with other agents or methods known to be standard care fortreating patients suffering from diseases or conditions characterized inpart by elevated blood glucose or ketone levels, such as, but notlimited to, diabetes. Such standard therapy may include fluidadministration, or administration of any other pharmaceutical agentsuseful for lowering blood glucose, ketones, or lipids, or for weightreduction.

The anti-hGCGR antibodies of the invention, or antigen-binding fragmentsthereof, may function to block the interaction between glucagon and itsreceptor, thereby inhibiting the glucose elevating effects of glucagon.The use of glucagon receptor antagonists, such as the antibodiesdescribed herein, may be an effective means of achieving normal levelsof glucose, thereby ameliorating, or preventing one or more symptoms of,or long term complications associated with, for example, diabetes. Theuse of glucagon receptor antagonists, such as the antibodies describedherein, may also be an effective means of achieving normal levels ofglucose in non-diabetic patients, who experience hyperglycemia as aresult of conditions or disorders not related to diabetes, such asperioperative hyperglycemia (hyperglycemia observed in patients justprior to surgery, or after surgery, discussed in greater detail below).In certain embodiments, methods of lowering blood glucose levels orketone levels in diabetic ketoacidosis are envisioned using theantibodies of the invention. In certain embodiments, methods of treatingpatients to achieve a reduction in body weight, or to prevent weightgain, or to maintain a normal body weight, are also envisioned using theantibodies of the invention.

The antibodies of the present invention, or antigen-binding fragmentsthereof, may be useful for ameliorating conditions such as, for example,impaired glucose tolerance, obesity, or for preventing weight gain, orfor treating diabetic conditions, or for preventing or reducing theseverity of any one or more of the long-term complications associatedwith diabetes, such as nephropathy, neuropathy, retinopathy, cataracts,stroke, atherosclerosis, impaired wound healing and other complicationsassociated with diabetes, known to those skilled in the art.

In a sixth aspect, the invention features a method for preventing theonset of stress hyperglycemia in a patient, or for treating a patientsuffering from stress hyperglycemia (also referred to as “stress-inducedhyperglycemia”), the method comprising administering to a patient atherapeutically effective amount of a composition comprising a glucagonreceptor antagonist, wherein the patient exhibits elevated levels ofblood glucose caused, or exacerbated by, one or more stress-inducingstimulus or one or more glucose elevating stimulus.

In one embodiment, the patient is identified on the basis of having ablood glucose level greater than about 140 mg/dL.

In one embodiment, the stress-inducing stimulus or the glucose elevatingstimulus is selected from the group consisting of: pre-existing type 1or type 2 diabetes; hypertonic dehydration; infusion of catecholaminepressors; glucocorticoid therapy; obesity; aging; excessive dextroseadministration; parenteral nutrition, enteral nutrition, pancreatitis;sepsis; stroke; traumatic head injury; hypothermia; hypoxemia; uremia;cirrhosis; anesthesia; pre-operative or post-operative hospital stays(peri-operative hyperglycemia); admission to an emergency room, a traumacenter, or an intensive care unit; prolonged hospital stays; surgicalprocedures; an infection; or a chronic illness.

In one embodiment, the glucagon receptor antagonist used to treat thestress hyperglycemia is an isolated human antibody, or an antigenbinding fragment thereof, specific for the human glucagon receptor,wherein the isolated human antibody or the antigen-binding fragmentthereof is capable of blocking the binding of glucagon to the glucagonreceptor.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof useful for treating stress hyperglycemia comprises thecomplementarity determining regions (CDRs) of a heavy chain variableregion (HCVR), wherein the HCVR has an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106,110, 126, 130 and 146; and the CDRs of a light chain variable region(LCVR), wherein the LCVR has an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108,118, 128, 138 and 148.

In a related embodiment, the isolated human antibody or antigen-bindingfragment thereof useful for treating stress hyperglycemia comprises aheavy chain variable region (HCVR) having an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 70,86, 90, 106, 110, 126, 130 and 146.

In another related embodiment, the isolated human antibody orantigen-binding fragment thereof useful for treating stresshyperglycemia comprises a light chain variable region (LCVR) having anamino acid sequence selected from the group consisting of SEQ ID NO: 10,26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138 and 148.

In another related embodiment, the isolated human antibody orantigen-binding fragment thereof useful for treating stresshyperglycemia comprises: (a) a HCVR having an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 70,86, 90, 106, 110, 126, 130 and 146; and (b) a LCVR having an amino acidsequence selected from the group consisting of SEQ ID NO: 10, 26, 42,58, 68, 78, 88, 98, 108, 118, 128, 138 and 148.

In another related embodiment, the isolated human antibody orantigen-binding fragment thereof useful for treating stresshyperglycemia comprises:

-   -   (a) a HCDR1 domain having an amino acid sequence selected from        the group consisting of SEQ ID NO: 4, 20, 36, 52, 72, 92, 112        and 132;    -   (b) a HCDR2 domain having an amino acid sequence selected from        the group consisting of SEQ ID NO: 6, 22, 38, 54, 74, 94, 114        and 134;    -   (c) a HCDR3 domain having an amino acid sequence selected from        the group consisting of SEQ ID NOs: 8, 24, 40, 56, 76, 96, 116        and 136;    -   (d) a LCDR1 domain having an amino acid sequence selected from        the group consisting of SEQ ID NO: 12, 28, 44, 60, 80, 100, 120        and 140;    -   (f) a LCDR2 domain having an amino acid sequence selected from        the group consisting of SEQ ID NO: 14, 30, 46, 62, 82, 102, 122        and 142; and    -   (g) a LCDR3 domain having an amino acid sequence selected from        the group consisting of SEQ ID NO: 16, 32, 48, 64, 84, 104, 124        and 144.

In one embodiment, the isolated human antibody or antigen-bindingfragment thereof useful for treating stress hyperglycemia comprises aHCVR/LCVR sequence pair selected from the group consisting of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/68, 70/78, 86/88, 90/98, 106/108, 110/118,126/128, 130/138, and 146/148.

In one embodiment, the patients who suffer from stress hyperglycemia whomay benefit by treatment with an antibody of the invention, as describedherein, may be diabetic patients or non-diabetic patients. In certainembodiments, these patients may be critically ill (requiring treatmentin an intensive care unit) or non-critically ill (not requiringtreatment in an intensive care unit) diabetic or non-diabetic patients.

In certain embodiments, the patients suffering from stress hyperglycemiawho are diabetic and who are candidates for therapy with an antibody ofthe invention may also receive treatment with one or more therapeuticagents selected from the group consisting of insulin, a biguanide(metformin), a sulfonylurea (such as glyburide, glipizide), a PPAR gammaagonist (pioglitazone, rosiglitazone), an alpha glucosidase inhibitor(acarbose, voglibose), SYMLIN® (pramlintide), any GLP-1 compound, or ananalogue, an agonist, a derivative, or a secretagogue thereof, and adipeptidyl peptidase 4 inhibitor.

In certain embodiments, the patients suffering from stress hyperglycemiawho are non-diabetic and who are candidates for therapy with an antibodyof the invention may also receive treatment with insulin.

In certain embodiments, the anti-GCGR antibodies, or antigen-bindingfragments thereof, useful for treating stress hyperglycemia, may lowerblood glucose levels in this patient population to levels within thenormal range. In one embodiment, the administering of an anti-GCGRantibody, or an antigen-binding fragment thereof, results in loweringthe blood glucose level to between about 80 mg/dL to about 180 mg/dL. Inone embodiment, the administering of an anti-GCGR antibody, or anantigen-binding fragment thereof, results in lowering the blood glucoselevel to between about 80 mg/dL to about 140 mg/dL. In anotherembodiment, the administering of an anti-GCGR antibody, or anantigen-binding fragment thereof, results in lowering the blood glucoselevel to between about 80 mg/dL to about 110 mg/dL. In anotherembodiment, the administering of an anti-GCGR antibody, or anantigen-binding fragment thereof, results in lowering the blood glucoselevel to between about 100 mg/dL to about 140 mg/dL.

In one embodiment, the use of an antibody of the invention alone, orwhen combined with a standard insulin treatment paradigm, may decreaseglucose levels without hypoglycemic risk in diabetic patients.

In one embodiment, the use of an antibody of the invention alone, orwhen combined with a standard insulin treatment paradigm, may decreaseglucose levels without hypoglycemic risk in stress hyperglycemicpatients.

In one embodiment, the use of an antibody of the invention, when usedalone, or when combined with a standard insulin treatment paradigm, mayresult in a decreased risk for infection, organ failure, or death instress hyperglycemic patients.

In one embodiment, the use of an antibody of the invention may reduceblood glucose levels after administration to a diabetic patient.

In one embodiment, the use of an antibody of the invention may reduceblood glucose levels after administration to a patient suffering fromstress hyperglycemia.

In one embodiment, the use of an antibody of the invention may result innormalization of blood glucose levels within a few days afteradministration in diabetic patients, or in patients suffering fromstress hyperglycemia.

In certain embodiments, the use of an antibody of the invention alone,or when combined with a standard insulin treatment paradigm, may beexpected to improve patient outcomes and/or decrease hospital costs incritically ill and non-critically ill stress hyperglycemic patients.

More particularly, the human anti-GCGR antibodies of the invention maylower blood glucose to levels ranging from about 80 mg/dL to about 180mg/dL, or from about 80 mg/dL to about 140 mg/dL, but may do so withoutthe risk of inducing hypoglycemia or wide fluctuations in glucose. Inone embodiment, patients experiencing stress hyperglycemia who maybenefit from treatment with the antibodies of the invention may bediabetic or non-diabetic patients. In one embodiment, patientsexperiencing stress hyperglycemia who may benefit from treatment withthe antibodies of the invention may be critically ill patients (diabeticor non-diabetic), requiring admission to an intensive care unit fortreatment. In one embodiment, patients experiencing stress hyperglycemiawho may benefit from treatment with the antibodies of the invention maybe non-critically ill patients (diabetic or non-diabetic), who do notrequire admission to an intensive care unit, but who may have anextended hospital stay (about five days or longer).

Other conditions or disorders treatable by the therapeutic methods ofthe invention include hyperosmolar hyperglycemia syndrome,hyperinsulinemia, the metabolic syndrome, insulin resistance syndrome,impaired fasting glucose, or hyperglycemia associated withhypercholesterolemia, hypertriglyceridemia, hyperlipidemia, and generaldyslipidemias.

The antibodies may also be useful for treating patients with inoperableglucagonoma (pancreatic endocrine tumor with or without necrolyticmigratory erythema and hyperglycemia).

In a seventh aspect, the invention provides a method of reducing theamount/dosage of insulin necessary to lower blood glucose levels towithin a normal range in a patient at risk for developing stresshyperglycemia, or in a patient suffering from stress hyperglycemia, themethod comprising administering an isolated human monoclonal antibodythat binds specifically to the glucagon receptor concomitantly withinsulin.

In one embodiment, the dosage of insulin may be reduced by about 10% toabout 95%, preferably by about 30% to about 95%, more preferably byabout 50% to about 90%, more preferably by about 75% to about 90% whenadministered concomitantly with an isolated human antibody that bindsspecifically to the glucagon receptor.

In one embodiment, the dosage of insulin may be reduced by about 90%when administered concomitantly with an isolated human monoclonalantibody that binds specifically to the glucagon receptor, wherein theisolated human monoclonal antibody that binds specifically to theglucagon receptor comprises a HCVR/LCVR amino acid sequence pair as setforth in SEQ ID NOs: 86/88.

In one embodiment, the antibody that demonstrates an insulin sparingeffect (i.e. the ability to aid in lowering blood glucose when used inconjunction with suboptimal insulin doses, for example, a reduction ininsulin dose that ranges from about 30% to 95% of the normal insulindose needed to lower blood glucose to within a normal range when used asstand alone therapy), comprises the complementarity determining regions(CDRs) of a heavy chain variable region (HCVR), wherein the HCVR has anamino acid sequence selected from the group consisting of SEQ ID NOs: 2,18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130 and 146; and the CDRs ofa light chain variable region (LCVR), wherein the LCVR has an amino acidsequence selected from the group consisting of SEQ ID NOs: 10, 26, 42,58, 68, 78, 88, 98, 108, 118, 128, 138 and 148.

In one embodiment, the isolated human monoclonal antibody orantigen-binding fragment thereof that demonstrates an insulin sparingeffect comprises a heavy chain variable region (HCVR) having an aminoacid sequence selected from the group consisting of SEQ ID NO: 2, 18,34, 50, 66, 70, 86, 90, 106, 110, 126, 130 and 146.

In one embodiment, the isolated human monoclonal antibody orantigen-binding fragment thereof that demonstrates an insulin sparingeffect comprises a light chain variable region (LCVR) having an aminoacid sequence selected from the group consisting of SEQ ID NO: 10, 26,42, 58, 68, 78, 88, 98, 108, 118, 128, 138 and 148.

In one embodiment, the isolated human monoclonal antibody orantigen-binding fragment thereof that demonstrates an insulin sparingeffect comprises: (a) a HCVR having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 70, 86, 90, 106,110, 126, 130 and 146; and (b) a LCVR having an amino acid sequenceselected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 68, 78,88, 98, 108, 118, 128, 138 and 148.

In one embodiment, the isolated human monoclonal antibody orantigen-binding fragment thereof that demonstrates an insulin sparingeffect comprises:

(a) a HCDR1 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, 20, 36, 52, 72, 92, 112 and 132;(b) a HCDR2 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 6, 22, 38, 54, 74, 94, 114 and 134;(c) a HCDR3 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 8, 24, 40, 56, 76, 96, 116 and 136;(d) a LCDR1 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 12, 28, 44, 60, 80, 100, 120 and 140;(e) a LCDR2 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 14, 30, 46, 62, 82, 102, 122 and 142; and(f) a LCDR3 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 16, 32, 48, 64, 84, 104, 124 and 144.

In one embodiment, the isolated human monoclonal antibody orantigen-binding fragment comprises a HCVR/LCVR sequence pair selectedfrom the group consisting of SEQ ID NO: 2/10, 18/26, 34/42, 50/58,66/68, 70/78, 86/88, 90/98, 106/108, 110/118, 126/128, 130/138, and146/148.

In one embodiment, the isolated human monoclonal antibody comprises aHCVR/LCVR amino acid sequence pair as set forth in SEQ ID NOs: 86/88.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the percent change in blood glucose levels in C57BL6 miceafter administration of H4H1327P (anti-GCGR antibody), and/or H1H316P(anti-PCSK9 antibody) when given alone or in combination. Control (Xwith solid line); H4H1327P at 3 mg/kg (▪ with solid line); H4H1327P at10 mg/kg (▴ with solid line); H1H316P at 10 mg/kg (

with solid line); H4H1327P at 3 mg/kg+H1H316P at 10 mg/kg (

with dashed line); H4H1327P at 10 mg/kg+H1H316P at 10 mg/kg (◯ withdashed lines).

FIG. 2 shows plasma LDL-C levels in C57BL6 mice after administration ofH4H1327P, and/or H1H316P when given alone or in combination. Control (Xwith solid line); H4H1327P at 3 mg/kg (▪ with solid line); H4H1327P at10 mg/kg (▴ with solid line); H1H316P at 10 mg/kg (

with solid line); H4H1327P at 3 mg/kg+H1H316P at 10 mg/kg (

with dashed line); H4H1327P at 10 mg/kg+H1H316P at 10 mg/kg (◯ withdashed lines).

FIG. 3 shows plasma HDL-C levels in C57BL6 mice after administration ofH4H1327P, and/or H1H316P when given alone or in combination. Control (Xwith solid line); H4H1327P at 3 mg/kg (▪ with solid line); H4H1327P at10 mg/kg (▴ with solid line); H1H316P at 10 mg/kg (

with solid line); H4H1327P at 3 mg/kg+H1H316P at 10 mg/kg (

with dashed line); H4H1327P at 10 mg/kg+H1H316P at 10 mg/kg (◯ withdashed lines).

FIG. 4 shows total plasma cholesterol levels in C57BL6 mice afteradministration of H4H1327P, and/or H1H316P when given alone or incombination. Control (X with solid line); H4H1327P at 3 mg/kg (▪ withsolid line); H4H1327P at 10 mg/kg (▴ with solid line); H1H316P at 10mg/kg (

with solid line); H4H1327P at 3 mg/kg+H1H316P at 10 mg/kg (

with dashed line); H4H1327P at 10 mg/kg+H1H316P at 10 mg/kg (◯ withdashed lines).

FIG. 5 shows plasma triglyceride levels in C57BL6 mice afteradministration of H4H1327P, and/or H1H316P when given alone or incombination. Control (X with solid line); H4H1327P at 3 mg/kg (▪ withsolid line); H4H1327P at 10 mg/kg (▴ with solid line); H1H316P at 10mg/kg (

with solid line); H4H1327P at 3 mg/kg+H1H316P at 10 mg/kg (

with dashed line); H4H1327P at 10 mg/kg+H1H316P at 10 mg/kg (◯ withdashed lines).

FIG. 6 shows hepatic triglyceride levels in C57BL6 mice afteradministration of H4H1327P, and/or H1H316P when given alone or incombination. Control (X); H4H1327P at 3 mg/kg (▪); H4H1327P at 10 mg/kg(▴); H1H316P at 10 mg/kg (

); H4H1327P at 3 mg/kg+H1H316P at 10 mg/kg (

); H4H1327P at 10 mg/kg+H1H316P at 10 mg/kg (◯).

FIG. 7 shows the blood glucose levels in a C57BL6 mouse model of stresshyperglycemia (see Saha, J K, Exptl. Biol. & Med. (2005), 230:777-784)after administration of a control antibody or an anti-GCGR antibody.Briefly, groups of C57BL6 mice received an intramuscular injection of acombination of ketamine (120 mg/kg) and xylazine (5 mg/kg). Thirtyminutes later, each group received intravenous injections of either anisotype control antibody (•), or an anti-GCGR antibody designatedH4H1327P (▪), each at a concentration of 10 mg/kg. Blood glucose wasmeasured at 0.5, 1.5, 3, 4.5, 6 and 24 hours after ketamine/xylazineadministration.

FIG. 8 shows the effect of H4H1327P on glucose lowering when used aloneor in combination with insulin in a model of stress hyperglycemia. (

) Isotype negative control antibody in water; (⋄) Isotype negativecontrol antibody plus insulin at 0.1 U/kg; (Δ) Isotype negative controlantibody plus insulin at 0.033 U/kg; (∇) Isotype negative controlantibody plus insulin at 0.01 U/kg; (□) H4H1327P in water; (◯) H4H1327Pplus insulin at 0.01 U/kg.

FIG. 9 shows an outline of a pharmacology study in diabetic cynomolgusmonkeys using H4H1327P mAb.

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, preferred methods and materials are now described. Allpublications mentioned herein are incorporated herein by reference intheir entirety.

DEFINITIONS

The “glucagon receptor”, also referred to herein as “GCGR”, belongs tothe G protein-coupled receptor class 2 family and consists of a longamino terminal extracellular domain (See SEQ ID NO: 158 for DNA encodingthe N-terminal extracellular domain and SEQ ID NO: 159 for the aminoacid sequence of the N-terminal extracellular domain), seventransmembrane segments, and an intracellular C-terminal domain (Jelineket al., Science 259: 1614-1616 (1993), Segre et al., Trends Endocrinol.Metab 4:309-314 (1993)). Glucagon receptors are notably expressed on thesurface of hepatocytes where they bind to glucagon and transduce thesignal provided thereby into the cell. Accordingly, the term “glucagonreceptor” also refers to one or more receptors that interactspecifically with glucagon to result in a biological signal. DNAsequences encoding glucagon receptors of rat and human origin have beenisolated and disclosed in the art (EP0658200B1). The murine andcynomolgus monkey homologues have also been isolated and sequenced(Burcelin, et al., Gene 164 (1995) 305-310); McNally et al., Peptides 25(2004) 1171-1178). As used herein, “glucagon receptor” and “GCGR” areused interchangeably. The expression “GCGR”, “hGCGR” or fragmentsthereof, as used herein, refers to the human GCGR protein or fragmentthereof, unless specified as being from a non-human species, e.g. “mouseGCGR”, “rat GCGR”, or “monkey GCGR”. Moreover, “GCGR,” or “hGCGR”, asused herein, refers to human GCGR having the nucleic acid sequence shownin SEQ ID NO: 157 and the amino acid sequence of SEQ ID NO: 153, or abiologically active fragment thereof. There are a variety of sequencesrelated to the GCGR gene having the following Genbank Accession Numbers:NP_(—)000151.1 (human), NP_(—)742089.1 (rat), XP_(—)001111894.1 (rhesusmonkey), and NP_(—)032127.2 (mouse). Other sequences disclosed hereininclude human GCGR (SEQ ID NO: 153), mouse GCGR (SEQ ID NO: 154),Cynomolgus monkey (SEQ ID NO: 155), rat GCGR (SEQ ID NO: 156). Incertain embodiments, fusion proteins useful in the invention may includeSEQ ID NO: 149 (hGCGR-hFc, residues 27-144 of NP_(—)000151.1 fused tothe Fc region of human IgG) SEQ ID NO:150 (hGCGR-hFc, residues 27-144 ofNP_(—)000151.1 fused to the Fc region of human IgG), SEQ ID NO:151(hGCGR-mmH, residues 27-144 of NP_(—)000151.1 fused to a myc-myc-histag), and SEQ ID NO:152 (MfGCGR-hFc, containing the N-terminal sequenceof Mf, cynomolgus monkey, which is identical to residues 27-144 of GCGRof the rhesus monkey, Macaca mulatta, having accession numberXP_(—)001111894.1, and which is fused to the Fc region of human IgG).The nucleic acid sequences, the polypeptides encoded by them, and othernucleic acid and polypeptide sequences are herein incorporated byreference in their entireties as well as for individual subsequencescontained therein.

The term “human proprotein convertase subtilisin/kexin type 9” or“hPCSK9”, as used herein, refers to hPCSK9 encoded by the nucleic acidsequence shown in SEQ ID NO:192 and having the amino acid sequence ofSEQ ID NO:193, or a biologically active fragment thereof.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds(i.e., “full antibody molecules”), as well as multimers thereof (e.g.IgM) or antigen-binding fragments thereof. Each heavy chain is comprisedof a heavy chain variable region (“HCVR” or “V_(H)”) and a heavy chainconstant region (comprised of domains C_(H)1, C_(H)2 and C_(H)3). Eachlight chain is comprised of a light chain variable region (“LCVR or“V_(L)”) and a light chain constant region (C_(L)). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, theFRs of the anti-GCGR antibody (or antigen binding fragment thereof) maybe identical to the human germline sequences, or may be naturally orartificially modified. An amino acid consensus sequence may be definedbased on a side-by-side analysis of two or more CDRs.

Substitution of one or more CDR residues or omission of one or more CDRsis also possible. Antibodies have been described in the scientificliterature in which one or two CDRs can be dispensed with for binding.Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regionsbetween antibodies and their antigens, based on published crystalstructures, and concluded that only about one fifth to one third of CDRresidues actually contact the antigen. Padlan also found many antibodiesin which one or two CDRs had no amino acids in contact with an antigen(see also, Vajdos et al. 2002 J Mol Biol 320:415-428).

CDR residues not contacting antigen can be identified based on previousstudies (for example residues H60-H65 in CDRH2 are often not required),from regions of Kabat CDRs lying outside Chothia CDRs, by molecularmodeling and/or empirically. If a CDR or residue(s) thereof is omitted,it is usually substituted with an amino acid occupying the correspondingposition in another human antibody sequence or a consensus of suchsequences. Positions for substitution within CDRs and amino acids tosubstitute can also be selected empirically. Empirical substitutions canbe conservative or non-conservative substitutions.

The fully-human anti-GCGR antibodies disclosed herein may comprise oneor more amino acid substitutions, insertions and/or deletions in theframework and/or CDR regions of the heavy and light chain variabledomains as compared to the corresponding germline sequences. Suchmutations can be readily ascertained by comparing the amino acidsequences disclosed herein to germline sequences available from, forexample, public antibody sequence databases. The present inventionincludes antibodies, and antigen-binding fragments thereof, which arederived from any of the amino acid sequences disclosed herein, whereinone or more amino acids within one or more framework and/or CDR regionsare mutated to the corresponding residue(s) of the germline sequencefrom which the antibody was derived, or to the corresponding residue(s)of another human germline sequence, or to a conservative amino acidsubstitution of the corresponding germline residue(s) (such sequencechanges are referred to herein collectively as “germline mutations”). Aperson of ordinary skill in the art, starting with the heavy and lightchain variable region sequences disclosed herein, can easily producenumerous antibodies and antigen-binding fragments which comprise one ormore individual germline mutations or combinations thereof. In certainembodiments, all of the framework and/or CDR residues within the V_(H)and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antibody was derived. In otherembodiments, only certain residues are mutated back to the originalgermline sequence, e.g., only the mutated residues found within thefirst 8 amino acids of FR1 or within the last 8 amino acids of FR4, oronly the mutated residues found within CDR1, CDR2 or CDR3. In otherembodiments, one or more of the framework and/or CDR residue(s) aremutated to the corresponding residue(s) of a different germline sequence(i.e., a germline sequence that is different from the germline sequencefrom which the antibody was originally derived). Furthermore, theantibodies of the present invention may contain any combination of twoor more germline mutations within the framework and/or CDR regions,e.g., wherein certain individual residues are mutated to thecorresponding residue of a particular germline sequence while certainother residues that differ from the original germline sequence aremaintained or are mutated to the corresponding residue of a differentgermline sequence. Once obtained, antibodies and antigen-bindingfragments that contain one or more germline mutations can be easilytested for one or more desired property such as, improved bindingspecificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes anti-hGCGR antibodies comprisingvariants of any of the HCVR, LCVR, and/or CDR amino acid sequencesdisclosed herein having one or more conservative substitutions. Forexample, the present invention includes anti-hGCGR antibodies havingHCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acidsubstitutions relative to any of the HCVR, LCVR, and/or CDR amino acidsequences disclosed herein.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human mAbs of the invention mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs and in particular CDR3. However, the term “human antibody”, as usedherein, is not intended to include mAbs in which CDR sequences derivedfrom the germline of another mammalian species (e.g., mouse), have beengrafted onto human FR sequences. The anti-human GCGR antibodies of theinvention may be designated as “anti-hGCGR” or “anti-GCGR”.

The term “specifically binds,” or the like, means that an antibody orantigen-binding fragment thereof forms a complex with an antigen that isrelatively stable under physiologic conditions. Specific binding can becharacterized by an equilibrium dissociation constant of at least about1×10⁻⁶ M or less (e.g., a smaller K_(D) denotes a tighter binding).Methods for determining whether two molecules specifically bind are wellknown in the art and include, for example, equilibrium dialysis, surfaceplasmon resonance, and the like. An isolated antibody that specificallybinds hGCGR may, however, exhibit cross-reactivity to other antigenssuch as GCGR molecules from other species. Moreover, multi-specificantibodies that bind to hGCGR and one or more additional antigens or abi-specific that binds to two different regions of hGCGR are nonethelessconsidered antibodies that “specifically bind” hGCGR, as used herein.

The term “high affinity” antibody refers to those mAbs having a bindingaffinity to hGCGR, expressed as K_(D), of at least 10⁻⁹ M; preferably10⁻¹⁰M; more preferably 10⁻¹¹ M, even more preferably 10⁻¹² M, asmeasured by surface plasmon resonance, e.g., BIACORE™ orsolution-affinity ELISA.

By the term “slow off rate”, “Koff” or “kd” is meant an antibody thatdissociates from hGCGR with a rate constant of 1×10⁻³ s⁻¹ or less,preferably 1×10⁻⁴s⁻¹ or less, as determined by surface plasmonresonance, e.g., BIACORE™.

The terms “antigen-binding portion” of an antibody, “antigen-bindingfragment” of an antibody, and the like, as used herein, include anynaturally occurring, enzymatically obtainable, synthetic, or geneticallyengineered polypeptide or glycoprotein that specifically binds anantigen to form a complex. The terms “antigen-binding portion” of anantibody, or “antibody fragment”, as used herein, refers to one or morefragments of an antibody that retain the ability to specifically bind tohGCGR.

The specific embodiments, antibody or antibody fragments of theinvention may be conjugated to a therapeutic moiety (“immunoconjugate”),such as a second GCGR antagonist, or to biguanide (metformin), asulfonylurea (such as glyburide, glipizide), a PPAR gamma agonist (suchas pioglitazone, or rosiglitazone), an alpha glucosidase inhibitor (suchas acarbose, or voglibose), EXENATIDE® (glucagon-like peptide 1),SYMLIN® (pramlintide), a chemotherapeutic agent, a radioisotope, or anyother therapeutic moiety useful for treating a disease or conditioncaused in part by unwanted glucagon activity.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies (Abs) havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds hGCGR, or a fragment thereof, is substantially freeof Abs that specifically bind antigens other than hGCGR).

A “blocking antibody” or a “neutralizing antibody”, as used herein (oran “antibody that neutralizes GCGR activity”), is intended to refer toan antibody whose binding to hGCGR results in inhibition of at least onebiological activity of GCGR. For example, an antibody of the inventionmay aid in preventing the increase in blood glucose levels associatedwith elevation of glucagon levels. Alternatively, an antibody of theinvention may demonstrate the ability to block cAMP production inresponse to glucagon. This inhibition of the biological activity of GCGRcan be assessed by measuring one or more indicators of GCGR biologicalactivity by one or more of several standard in vitro or in vivo assaysknown in the art (see examples below).

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-timebiomolecular interactions by detection of alterations in proteinconcentrations within a biosensor matrix, for example using the BIACORE™system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. The term“epitope” also refers to a site on an antigen to which B and/or T cellsrespond. It also refers to a region of an antigen that is bound by anantibody. Epitopes may be defined as structural or functional.Functional epitopes are generally a subset of the structural epitopesand have those residues that directly contribute to the affinity of theinteraction. Epitopes may also be conformational, that is, composed ofnon-linear amino acids. In certain embodiments, epitopes may includedeterminants that are chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl groups, or sulfonylgroups, and, in certain embodiments, may have specific three-dimensionalstructural characteristics, and/or specific charge characteristics.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 90%, and more preferablyat least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or GAP, as discussed below. A nucleic acid molecule havingsubstantial identity to a reference nucleic acid molecule may, incertain instances, encode a polypeptide having the same or substantiallysimilar amino acid sequence as the polypeptide encoded by the referencenucleic acid molecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 90% sequence identity, even more preferably atleast 95%, 98% or 99% sequence identity. Preferably, residue positions,which are not identical, differ by conservative amino acidsubstitutions. A “conservative amino acid substitution” is one in whichan amino acid residue is substituted by another amino acid residuehaving a side chain (R group) with similar chemical properties (e.g.,charge or hydrophobicity). In general, a conservative amino acidsubstitution will not substantially change the functional properties ofa protein. In cases where two or more amino acid sequences differ fromeach other by conservative substitutions, the percent or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment are wellknown to those of skill in the art. See, e.g., Pearson (1994) MethodsMol. Biol. 24: 307-331, which is herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include 1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains:serine and threonine; 3) amide-containing side chains: asparagine andglutamine; 4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; 5) basic side chains: lysine, arginine, and histidine; 6)acidic side chains: aspartate and glutamate, and 7) sulfur-containingside chains: cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443 45, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides is typically measured usingsequence analysis software. Protein analysis software matches similarsequences using measures of similarity assigned to varioussubstitutions, deletions and other modifications, including conservativeamino acid substitutions. For instance, GCG software contains programssuch as GAP and BESTFIT which can be used with default parameters todetermine sequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and a mutein thereof. See,e.g., GCG Version 6.1. Polypeptide sequences also can be compared usingFASTA with default or recommended parameters; a program in GCG Version6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percentsequence identity of the regions of the best overlap between the queryand search sequences (Pearson (2000) supra). Another preferred algorithmwhen comparing a sequence of the invention to a database containing alarge number of sequences from different organisms is the computerprogram BLAST, especially BLASTP or TBLASTN, using default parameters.See, e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403 410 and (1997)Nucleic Acids Res. 25:3389 402, each of which is herein incorporated byreference.

In specific embodiments, the antibody or antibody fragment for use inthe method of the invention may be mono-specific, bi-specific, ormulti-specific. Multi-specific antibodies may be specific for differentepitopes of one target polypeptide or may contain antigen-bindingdomains specific for epitopes of more than one target polypeptide. Anexemplary bi-specific antibody format that can be used in the context ofthe present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bi-specific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise an Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1 mAbs;N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the caseof IgG2 mAbs; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT;Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the caseof IgG4 mAbs. Variations on the bi-specific antibody format describedabove are contemplated within the scope of the present invention.

By the phrase “therapeutically effective amount” is meant an amount thatproduces the desired effect for which it is administered. The exactamount will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques (see, forexample, Lloyd (1999) The Art, Science and Technology of PharmaceuticalCompounding).

The term “blood glucose level”, or “level of blood glucose” shall meanblood glucose concentration. In certain embodiments, a blood glucoselevel is a plasma glucose level. Plasma glucose may be determined inaccordance with Etgen et al., (Metabolism 2000; 49(5): 684-688) orcalculated from a conversion of whole blood glucose concentration inaccordance with D'Orazio et al., (Clin. Chem. Lab. Med. 2006; 44(12):1486-1490).

“Normal glucose levels” refers to mean plasma glucose values in humansof less than about 100 mg/dL for fasting levels, and less than about 145mg/dL for 2-hour post-prandial levels or 125 mg/dL for a random glucose.

The term “elevated blood glucose level” or “elevated levels of bloodglucose” shall mean an elevated blood glucose level such as that foundin a subject demonstrating clinically inappropriate basal andpostprandial hyperglycemia or such as that found in a subject in oralglucose tolerance test (oGTT), with “elevated levels of blood glucose”being greater than about 100 mg/dL when tested under fasting conditions,and greater than about 200 mg/dL when tested at 1 hour.

“Cholesterol normalization” or “normal cholesterol levels” refers to atotal cholesterol level in a human of about less than 200 mg/dL, with arange of about 200-240 mg/dL considered borderline high. From the totalnormal cholesterol, a mean LDL value in humans of about 100 to about 129mg/dL is considered normal and an HDL value above 45 mg/dL is considerednormal. The normal triglyceride level in humans is less than 150 mg/dL.The normal total/HDL ratio is below 4.5, and the normal LDL/HDL ratio isless than 3. These values may be determined in accordance with standardlaboratory practice (see also, Friedewald, W T, Clin. Chem. (1972),18:499-502; Chen, Y. et al. Lipids Health Dis. (2010); 9:52; Keevil, JG, et al., Circulation (2007), 115:1363-1370; and Bairaktari, E. et al.,Clin. Biochem. (2000), 33:549-555). In certain embodiments of theinvention, the anti-GCGR antibodies may be useful to lower blood glucoselevels to within the normal range. In certain embodiments of theinvention, the anti-GCGR antibodies may be useful to increase the levelof HDL-C. In certain embodiments of the invention, the anti-GCGRantibodies may be useful to decrease the level of triglycerides.

The term “glucagon receptor antagonist” refers to any molecule, eithernatural, or synthetic, including proteins, peptides, nucleic acids,antibodies, or small organic molecules. A “glucagon receptorantagonist”, as described herein, may inhibit the binding of glucagon toits receptor, thereby preventing at least one biological activityassociated with the binding of glucagon to its receptor, e.g. elevationof blood glucose levels. Examples of glucagon receptor antagonists aredescribed in, for example, U.S. Pat. Nos. 7,947,809; 8,158,759;7,989,472 and 7,494,978, as well as in the following US patentpublications: US2008/036341, US2011/0223160, US2009/0041784,US2011/0312911 and in the following WO publications: WO2007/120284,WO2011/037815 and WO2011/119541.

The term “stress hyperglycemia”, which is used interchangeably with“stress-induced hyperglycemia”, refers to a condition whereby a patientsuffers from a transient increase in blood glucose (>140 mg/dL) that istemporally linked to the stress of an acute injury or illness. Stresshyperglycemia can occur in patients with or without a history ofdiabetes. The cause is thought to be directly related to the stress ofthe underlying medical illness, anesthesia, surgery, or trauma.

Stress hyperglycemia is the result of, or may be exacerbated by, any oneor more of the following risk factors, conditions or therapies:pre-existing type 1 or type 2 diabetes; hypertonic dehydration; infusionof catecholamine pressors; glucocorticoid therapy; obesity; aging;excessive dextrose administration; parenteral nutrition, enteralnutrition, pancreatitis; sepsis; stroke; traumatic head injury;hypothermia; hypoxemia; uremia; cirrhosis; anesthesia; pre-operative orpost-operative hospital stays (peri-operative hyperglycemia); admissionto an emergency room, a trauma center, or an intensive care unit;prolonged hospital stays; surgical procedures; an infection; or aworsening chronic illness.

The term “critically ill”, as used herein, generally refers to a patientsuffering from a disease, disorder, injury, surgical procedure, or othercondition who requires treatment or monitoring in a critical care unit,or an intensive care unit of a hospital. In its broadest sense, the terma “critically ill” patient, as used herein refers to a patient who hassustained or is at risk of sustaining acutely life-threatening single ormultiple organ system failure. A critically ill patient may be a“diabetic patient”, e.g. a patient having been diagnosed as havingdiabetes using standard tests known to those skilled in the art; or a“non-diabetic patient”, e.g. a patient who has been diagnosed as nothaving diabetes using standard methods known to those skilled in theart.

The term “not-critically ill”, or “non-critically ill” refers to ahospitalized patient suffering from a disease, disorder, or conditionthat does not require treatment or monitoring in a critical care unit,or intensive care unit of a hospital. In its broadest sense, the term a“not-critically ill” patient, as used herein refers to a patient otherthan one who has sustained or is at risk of sustaining acutelylife-threatening single or multiple organ system failure due to disease,injury, surgical procedure, or other condition.

The term “Intensive Care Unit” (herein designated ICU), as used hereinrefers to the part of a hospital where critically ill patients aretreated. This might vary from country to country and even from hospitalto hospital and this part of the hospital may not necessary, officially,bear the name “Intensive Care Unit” or a translation or derivationthereof. The term “Intensive Care Unit” also covers any health care unitthat treats patients with life-threatening conditions requiringconstant, close monitoring and support from equipment and medication inorder to maintain normal bodily functions.

The term “treating” or “treatment”, as used herein, refers to anapproach for obtaining beneficial or desired clinical results. Forpurposes of this invention, beneficial or desired clinical resultsinclude, but are not limited to, one or more of the following:improvement in blood glucose to within about 80-180 mg/dL, or to withinabout 80-140 mg/dL, or an improvement in any one or more conditions,diseases, or symptoms associated with, or resulting from, elevatedlevels of blood glucose, including, but not limited to susceptibility toinfections, organ failure, disability after stroke, polyneuropathy,arrhythmia, or mortality in patients. In addition, “treating” with aglucagon receptor antagonist of the invention may result in a beneficialor desired clinical result which may include an improvement in bloodglucose level to within about 80-180 mg/dL, or to within about 80-140mg/dL, in any condition or disease resulting from exposure to any one ormore stress-inducing stimulus or glucose elevating stimulus selectedfrom the group consisting of: pre-existing type 1 or type 2 diabetes;infusion of catecholamine pressors; parenteral nutrition; enteralnutrition; glucocorticoid therapy; obesity; aging; excessive dextroseadministration; pancreatitis; sepsis; stroke; traumatic head injury;hypothermia; hypoxemia; uremia; cirrhosis; anesthesia; pre-operative orpost-operative hospital stays (peri-operative hyperglycemia); admissionto an emergency room, a trauma center, or an intensive care unit;prolonged hospital stays; surgical procedures; an infection; and achronic illness. “Treating” with a glucagon receptor antagonist of theinvention may also lead to prevention of the onset of stresshyperglycemia.

A “stress-inducing stimulus”, which is used interchangeably with a“glucose-elevating stimulus”, refers to an event that promotes theelevation of blood glucose to above-normal levels. Examples of a“stress-inducing stimulus”, or a “glucose-elevating stimulus” includeany one or more of the following: pre-existing type 1 or type 2diabetes; hypertonic dehydration; infusion of catecholamine pressors;parenteral nutrition; enteral nutrition; glucocorticoid therapy;obesity; aging; excessive dextrose administration; pancreatitis; sepsis;stroke; traumatic head injury; hypothermia; hypoxemia; uremia;cirrhosis; anesthesia; pre-operative or post-operative hospital stays(pen-operative hyperglycemia); admission to an emergency room, a traumacenter, or an intensive care unit; prolonged hospital stays; surgicalprocedures; an infection; and a chronic illness.

The term “insulin”, as used herein refers to insulin from any speciessuch as human insulin, porcine insulin, bovine insulin and saltsthereof, such as zinc salts.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone derived from theposttranslational modification of proglucagon and secreted by gutendocrine cells. GLP-1 mediates its actions through a specific Gprotein-coupled receptor (GPCR), namely GLP-1R. GLP-1 is bestcharacterized as a hormone that regulates glucose homeostasis. GLP-1 hasbeen shown to stimulate glucose-dependent insulin secretion and toincrease pancreatic beta cell mass. GLP-1 has also been shown to reducethe rate of gastric emptying and to promote satiety. The efficacy ofGLP-1 peptide agonists in controlling blood glucose in Type 2 diabeticshas been demonstrated in several clinical studies [see, e.g., Nauck etal., Drug News Perspect (2003) 16:413-422], as has its efficacy inreducing body mass [Zander et al., Lancet (2002) 359:824-830].

The term “GLP-1 compound”, as used herein refers to GLP-1(1-37),exendin-4(1-39), insulinotropic (insulin stimulating) fragments thereof,insulinotropic analogs thereof and insulinotropic derivatives thereof.Insulinotropic fragments of GLP-1(1-37) are insulinotropic peptides forwhich the entire sequence can be found in the sequence of GLP-1(1-37)and where at least one terminal amino acid has been deleted. Examples ofinsulinotropic fragments of GLP-1(1-37) are GLP-1(7-37) wherein theamino acid residues in positions 1-6 of GLP-1(1-37) have been deleted,and GLP-1(7-36) where the amino acid residues in position 1-6 and 37 ofGLP-1(1-37) have been deleted. Insulinotropic derivatives of GLP-1(1-37)and analogs thereof are what the person skilled in the art may considerto be derivatives of these peptides, i.e. having at least onesubstituent which is not present in the parent peptide molecule with theproviso that said derivative either is insulinotropic or is a prodrug ofan insulinotropic compound. Examples of substituents are amides,carbohydrates, alkyl groups and lipophilic substituents. Furtherexamples of GLP-1(1-37) insulinotropic fragments thereof, insulinotropicanalogs thereof and insulinotropic derivatives thereof are described inWO 98/08871, WO 99/43706, U.S. Pat. No. 5,424,286 and WO 00/09666.

The term “GLP-1 agonist”, as used herein refers to a molecule,preferably GLP-1 or an analogue or a derivative thereof, or exendin oran analogue or a derivative thereof, or a non-peptidyl compound, whichinteracts with the GLP-1 receptor and induces the physiological andpharmacological characteristics of the GLP-1 receptor. Methods foridentifying GLP-1 agonists are described in WO 93/19175. The term “GLP-1agonist” is also intended to comprise active metabolites and prodrugsthereof, such as active metabolites and prodrugs of GLP-1 or an analogueor a derivative thereof, or exendin or an analogue or a derivativethereof, or a non-peptidyl compound. A “metabolite” is an activederivative of a GLP-1 agonist produced when the GLP-1 agonist ismetabolized. A “prodrug” is a compound which is either metabolized to aGLP-1 agonist or is metabolized to the same metabolite(s) as a GLP-1agonist.

The term “GLP-1 secretagogue” shall mean an agent (e.g., a compound)that promotes GLP-1 secretion from a cell, e.g. an enteroendocrine cell.

The term “dipeptidyl-peptidase IV inhibitor” or “DPP-IV inhibitor,” asused herein, refers to a compound that binds to DPP-IV and inhibitsDPP-IV dipeptidyl peptidase activity. Dipeptidyl peptidase IV exhibitscatalytic activity against a broad range of peptide substrates thatincludes peptide hormones, neuropeptides, and chemokines. The incretinsglucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropicpolypeptide (GIP), which stimulate glucose-dependent insulin secretionand otherwise promote blood glucose homeostasis, are rapidly cleaved byDPP-IV at the position 2 alanine leading to inactivation of theirbiological activity. Both pharmacological and genetic attenuation ofDPP-IV activity is associated with enhanced incretin action, increasedinsulin, and lower blood glucose in vivo. A second-generation DPP-IVinhibitor, LAF237 (Ahren et al., J Clin Endocrinol Metab (2004)89:2078-2084; and Villhauer et al., J Med Chem (2003) 46:2774-2789), iscurrently in phase 3 clinical trials for Type 2 diabetes and additionalDPP-IV inhibitors are in various stages of clinical development,including MK-0431 (sitagliptin), BMS-477118 (saxagliptin), Onglyza,Linagliptin, PSN-9301, SYR-322 and SYR-619, Vildagliptin, Alogliptin,R1438, TA-6666, PHX1149, GRC 8200, TS-021, SSR 162369, and ALS 2-0426.

GENERAL DESCRIPTION

The therapeutic value of glycemic control is well documented. However, asubstantial unmet need remains for agents that provide appropriateglycemic control with a low risk of hypoglycemia and a favorable sideeffect profile in both chronic and acute settings. The present inventionis directed to such agents. More particularly, the present inventionrelates to the use of glucagon receptor antagonists, more particularly,antibodies specific for the human glucagon receptor (GCGR), which inducerapid, potent and sustained glucose and ketone lowering in preclinicalmodels of hyperglycemia without evidence of hypoglycemia. Furthermore,in obese mouse models, the antibodies of the invention provide forrobust weight loss and body fat loss. Moreover, in an animal model ofstress hyperglycemia, the antibodies of the present inventiondemonstrate a significant effect on lowering blood glucose levels towithin a normal range.

Since glucagon exerts its physiological effects by signaling through theglucagon receptor, the glucagon receptor may be a potential therapeutictarget for diabetes and other glucagon related metabolic disorders. Theuse of glucagon receptor antagonists, such as the antibodies describedherein, may be an effective means of achieving normal levels of glucose,thereby ameliorating, or preventing one or more symptoms or long termcomplications associated with diabetes. The antibodies of the presentinvention may also be useful for ameliorating conditions associatedwith, for example, impaired glucose tolerance, for treating obesity, forpreventing weight gain, for treating metabolic syndrome, for treatinghyperglycemia, or for treating diabetic conditions, including diabeticketoacidosis, or for preventing and/or lowering the risk of developingany one or more of the complications associated with diabetes, such asnephropathy, neuropathy, retinopathy, cataracts, stroke,atherosclerosis, impaired wound healing and other complicationsassociated with diabetes, known to those skilled in the art.

The use of the anti-hGCGR antibodies, as described herein, may also beuseful for treating other conditions, including stress hyperglycemia(also known as stress-induced hyperglycemia, discussed in more detailbelow), hyperglycemic hyperosmolar syndrome (Stoner, G. D., AmericanFamily Physician, (2005), 71(9):1723-1730; Diabetes Spectrum, Umpierrez,G. E., (2002), 15(1):28-36; Nugent, B. W., Emergency Medicine Clinics ofNorth America, (2005), 23:629-648), perioperative hyperglycemia (Frisch,A. et al. Diabetes Care, (2010), 33(8):1783-1788; Hanazaki, K. et al.World J Gastroenterol, (2009), 15(33): 4122-4125; Smiley, D. D. et al.Southern Medical Journal, (2006), 99(6):580-589; Hermanides, J. et al.,The Netherlands J. of Med. 67(6):226-229; Maerz, L. L. et al., CurrentOpinion in Critical Care, (2011), 17:370-375), hyperglycemia inintensive care unit patients (Gunst, J. et al., Seminars in Dialysis,(2010), 23(2):157-162; Losser, M-R., Critical care, (2010), 14:231),hyperinsulinemia, and insulin resistance syndrome and glucagonoma(pancreatic endocrine tumor with or without necrolytic migratoryerythema and hyperglycemia) (See for example, Boden, G. et al., N Engl JMed (1986); 314:1686-1689).

In certain embodiments, the antibodies of the invention were obtainedfrom mice immunized with a primary immunogen, followed by immunizationwith a secondary immunogen. The immunogen may be a cell line expressingthe GCGR protein, or a biologically active fragment thereof, or DNAencoding the GCGR protein or active fragment thereof, or the GCGRprotein or active fragment thereof. For example, in certain embodiments,the primary immunogen may be a cell line engineered using standardprocedures known in the art to over-express full-length hGCGR (e.g. themouse MG87 cell line). Alternatively, DNA immunization may be performedusing DNA encoding full-length hGCGR (e.g. hGCGR constructs derived fromaccession number NP_(—)000151.1), or DNA encoding a biologically activefragment thereof, for example, DNA encoding the N-terminal domain ofGCGR (see, for example, SEQ ID NO: 158, which encodes SEQ ID NO: 159),or a soluble N-terminal protein, including that of SEQ ID NO: 159, orthe amino acids spanning residues 27-144 of SEQ ID NO: 153. Thesecondary immunogen may be a GCGR protein, or biologically activefragment thereof, or a fusion protein, such as hGCGR-mmH (REGN547, SEQID NO: 151) or hGCGR-hFc (REGN315, SEQ ID NO: 150; REGN316, SEQ ID NO:149).

In certain embodiments of the present invention, the N-terminal domain,having the amino acid sequence shown in SEQ ID NO: 159 (without thesignal sequence), or any one or more of the ectodomains of GCGR, e.g.any one or more of the extracellular regions (or fragments thereof) maybe used to prepare antibodies that bind GCGR and inhibit its function,e.g. its ability to bind glucagon, which would result in lowering ofblood glucose levels.

The full-length amino acid sequence of human GCGR is shown as SEQ ID NO:153. The signal peptide spans amino acid residues 1-26 of SEQ ID NO:153; the N-terminal domain spans residues 27-144 of SEQ ID NO: 153;extracellular region 1 (EC1) spans amino acid residues 194-226 of SEQ IDNO: 153; extracellular region 2 (EC2) spans amino acid residues 285-305of SEQ ID NO: 153; and extracellular region 3 (EC3) spans amino acidresidues 369-384 of SEQ ID NO:153.

In certain embodiments, antibodies that bind specifically to GCGR may beprepared using fragments of the above-noted extracellular regions, orpeptides that extend beyond the designated regions by about 5 to about20 amino acid residues from either, or both, the N or C terminal ends ofthe regions described herein. In certain embodiments, any combination ofthe above-noted regions or fragments thereof may be used in thepreparation of GCGR specific antibodies. In certain embodiments, any oneor more of the above-noted regions of GCGR, or fragments thereof may beused for preparing monospecific, bispecific, or multispecificantibodies.

Stress Hyperglycemia

Stress hyperglycemia is defined as a transient increase in blood glucose(>140 mg/dL) that is temporally linked to the stress of an acute injuryor illness. This common medical condition occurs frequently in thediabetic and non-diabetic hospitalized patient and requires prompttherapeutic intervention (Kitabchi A E, Umpierrez G E, Miles J M, FisherJ N, Diabetes Care, (2009), July; 32(7):1335-43; Baker E H, Janaway C H,Philips B J, Brennan A L, Baines D L, Wood D M, et al., Thorax, (2006),April; 61(4):284-9). Serious medical and surgical conditions associatedwith stress hyperglycemia include myocardial infarction, burns, andcardiopulmonary bypass surgery (Rocha D M, Santeusanio F, Faloona G R,Unger R H, N Engl J. Med. (1973), Apr 5; 288(14):700-3). Stresshyperglycemia also occurs in non-critically ill patients, for example,in those admitted for exacerbation of chronic obstructive pulmonarydisease (Oshima C, Kaneko T, Tsuruta R, Oda Y, Miyauchi T, Fujita M, etal., Resuscitation, (2010), February; 81(2):187-92). In hospitalizedpatients, stress hyperglycemia is associated with substantial increasesin infections, dialysis, blood transfusions, polyneuropathy, and othermedical complications including death (up to 18-fold).

Stress Hyperglycemia has increasingly been the focus of clinicalinvestigations that demonstrate reductions in the aforementioned medicalcomplications with intensive insulin therapy. However, intensive insulintherapy is associated with a substantial risk of hypoglycemia, whichgreatly reduces the medical benefits of therapy. As a result, currentpractice guidelines have been revised to target higher blood glucoselevels in order to lower the risk of hypoglycemia. This may limit thebenefit of therapy (Kitabchi A E, Umpierrez G E, Miles J M, Fisher J N,Diabetes Care, (2009), July; 32(7):1335-43; Baker E H, Janaway C H,Philips B J, Brennan A L, Baines D L, Wood D M, et al., Thorax, (2006),April; 61(4):284-9).

Studies in patients admitted for conditions associated with stresshyperglycemia have demonstrated sustained glucagon elevations up to10-fold above the normal levels (Rocha D M, Santeusanio F, Faloona G R,Unger R H, N Engl J. Med. (1973), Apr. 5; 288(14):700-3; Oshima C,Kaneko T, Tsuruta R, Oda Y, Miyauchi T, Fujita M, et al, Resuscitation,(2010), February; 81(2):187-92). In these settings, elevated glucagonmay contribute to elevated blood glucose levels and may hamper achievingtarget glycemic control. Therefore, adding a glucagon receptorantagonist, such as any one of the antibodies described herein, to thecurrent therapeutic regimen for stress hyperglycemia may reduce the riskof hypoglycemic episodes, allow patients to be treated to morephysiologic blood glucose levels, and reduce morbidity and mortality.The present invention describes the use of antibodies specific for theglucagon receptor for the short-term treatment of metabolicperturbations in patients with stress hyperglycemia.

Stress hyperglycemia is a common observation in many serious medicalconditions such as myocardial infarction, burns, and cardiopulmonarybypass surgery (Kitabchi A E, Umpierrez G E, Miles J M, Fisher J N,Diabetes Care, (2009), July; 32(7):1335-43). The prevalence of stresshyperglycemia has been estimated to occur in 32-38% of hospitalizedpatients; 41% of the critically ill; 44% with heart failure; and 80%post-cardiac surgery. Further, 33% of non-ICU patients with stresshyperglycemia and 80% of ICU patients with stress hyperglycemia have nohistory of DM before hospital admission (Umpierrez G E, Isaacs S D,Bazargan N, You X, Thaler L M, Kitabchi A E, J Clin Endocrinol Metab,(2002), March 87(3):978-82). In a single center study, it was determinedthat a large majority of patients (75%) seen in the cardiac emergencyunit with acute myocardial infarction had blood glucose levels ≧180mg/dL (Lerario A C, Coretti F M, Oliveira S F, Betti R T, Bastos Mdo S,Fern Lde A, et al., Arq Bras Endocrinol Metabol. (2008), April;52(3):465-72). This association of hyperglycemia in acutely ill patientshas been recognized for some time. In fact, hyperglycemia was includedin the predictive APACHE III severity of illness scoring system in 1991(Knaus W A, Wagner D P, Draper E A, Zimmerman J E, Bergner M, Bastos PG, et al. Chest, (1991) December; 100(6):1619-36). Hermanides et al.noted that immediately after induction of anesthesia, plasma glucoselevels started to increase until the second day post-operatively and didnot return to baseline until seven weeks (Hermanides, J. et al.Netherlands J. Med. (2009), Vol. 67(6):226-229).

Any of the fully human monoclonal antibodies described herein asglucagon receptor antagonists may be used for preventing the onset ofstress hyperglycemia by administering the antibody prior to an eventthat is stress-inducing, for example, prior to surgery. The antibodiesdescribed herein may also be administered therapeutically for treatingstress hyperglycemia, more particularly, for the treatment of metabolicperturbations in patients who are diagnosed with stress hyperglycemiaduring hospitalization. Accordingly, both prophylactic and therapeuticadministration of the anti-GCGR antibodies of the invention isenvisioned. One of the fully human monoclonal antibodies will bedeveloped for use in critically ill intensive care unit patients who areat high risk for death. In addition, one of the fully human monoclonalantibodies will be developed for use in non-critically ill patients whoare at a low risk for death, but who will benefit from improvedmetabolic control and thus experience lower morbidity. Currently, thereare no therapies specifically approved to prevent the onset of, or totreat stress hyperglycemia. Stress hyperglycemia in patients who arecritically ill represents a serious unmet medical need because: 1) thesepatients have a high risk for imminent death, 2) existing therapies,i.e., intensive insulin may paradoxically increase the risk for death bycausing severe hypoglycemia, and 3) the etiology of stress hyperglycemiais multifactorial, and insulin therapy alone may be insufficient foroptimal control. Therefore, to address this unmet medical need, theantibodies of the present invention will be tested further in patientseither suffering from stress hyperglycemia, or at risk for developingstress hyperglycemia (but prior to the onset of stress hyperglycemia).Recognized surrogates for mortality, such as improved glycemic controland improvement in the Acute Physiology and Chronic Health Evaluation(APACHE) score (Knaus W A, et al., “APACHE II: A Severity of DiseaseClassification System”, Critical Care Medicine, (1985), vol. 13, No. 10,pp. 818-829), will be used in the studies proposed herein.

Mortality in Stress Hyperglycemia

In ICU and non-ICU patients, elevated blood glucose levels areassociated with increased morbidity and mortality (Umpierrez G E, IsaacsS D, Bazargan N, You X, Thaler L M, Kitabchi A E, J Clin EndocrinolMetab. (2002), March 87(3):978-82; McAlister F A, Majumdar S R, Blitz S,Rowe B H, Romney J, Marrie T J, Diabetes Care. (2005), Apr.28(4):810-5). There are a number of mechanisms by which hyperglycemiamay adversely affect the outcome of the critically ill andnon-critically ill patient. In particular, hyperglycemia impairs whiteblood cell function resulting in abnormal granulocyte adhesion,phagocytosis, respiratory burst and superoxide formation, andintracellular killing (Rubinstein R, Genaro A M, Motta A, Cremaschi G,Wald M R, Clin Exp Immunol. (2008) November 154:235-46; Stegenga M E,van der Crabben S N, Dessing M C, Pater J M, van den Pangaart P S, deVos A F, et al., Diabet Med. (2008), February; 25(2):157-64). The riskof developing multiple organ failure increases and survival decreases ifinfection occurs (McMahon M M, Bistrian B R. Infect Dis Clin North Am.(1995) March 9(1):1-9; Capuano F, Roscitano A, Simon C, Sclafani G,Benedetto U, Comito C, et al. Heart Int. (2006), 2(1):49). In addition,ICU patients develop insulin resistance, in part due to an increase inglucagon. The hepatic insulin resistance leads to an increase in serumconcentrations of IGF-1 binding protein. IGF-1 binding protein has beenlinked to an increase risk of hospital death, possibly reflectingnegative effects on IGF-1 and unwanted effects on muscle wasting in thecritically ill patient (Van den Berghe G, Baxter R C, Weekers F, WoutersP, Bowers C Y, Veldhuis J D, J Clin Endocrinol Metab, (2000), January85(1): 183-92; Van den Berghe G, Wouters P, Weekers F, Mohan S, Baxter RC, Veldhuis J D, et al., J Clin Endocrinol Metab. (1999), April; 84(4):1311-23). Control of blood glucose has also been linked to a decrease inthe risk of critical illness polyneuropathy, either directly or throughdecreased infection and sepsis (Van den Berghe G, Wouters P, Weekers F,Verwaest C, Bruyninckx F, Schetz M, et al. N Engl J. Med. (2001), Nov.8; 345(19): 1359-67).

Under stress conditions, endogenous glucagon secretion is thought to beelevated by hypovolemia, hypoglycemia and low insulin levels (Young A,Adv Pharmacol. (2005), 52:151-71). Several studies in patients admittedto the hospital for acute myocardial infarction, coronary artery bypassgrafts, valve replacement, burns, or sepsis demonstrated glucagon levelselevated 2-10-fold above normal throughout the acute medical illness(Ellger B, Debaveye Y, Vanhorebeek I, Langouche L, Giulietti A, VanEtten E, et al. Diabetes. (2006), April; 55(4): 1096-105; Nygren J,Sammann M, Malm M, Efendic S, Hall K, Brismar K, et al. Clin Endocrinol(Oxf). (1995), October; 43(4): 491-500; Ascione R, Rogers C A,Rajakaruna C, Angelini G D, Circulation. (2008), Jul. 8; 118(2): 113-23;Jakob S M, Stanga Z. Nutrition. (2010), April; 26(4): 349-53. Therefore,it would prove beneficial to prevent the onset of stress hyperglycemiaby administering at least one antibody of the invention prophylacticallyto patients at risk for developing stress hyperglycemia. Alternatively,if the patient exhibits hyperglycemia due to exposure to a stressor, itwould be beneficial to administer the antibody therapeutically after theelevation of glucose resulting from exposure to the stressor. Theantibody may be administered as stand-alone therapy, or may beadministered as adjunct therapy with another glucose lowering agent, forexample, insulin.

Current Treatment of Stress Hyperglycemia

Treatment of stress hyperglycemia, currently limited primarily to theadministration of insulin, results in improved outcome. This concept issupported by a series of randomized controlled trials that examined theeffect of intensive insulin treatment in hospitalized patients anddemonstrated improved short-term outcome (reviewed in Krinsley J S,Meyfroidt G, van den Berghe G, Egi M, Bellomo R. Curr Opin Clin NutrMetab Care. (2012), March; 15(2): 151-60). In 2012, practice guidelinesfor treatment of stress hyperglycemia in critically ill andnon-critically ill patients were established and involved a consensusprocess including: The Endocrine Society members, American DiabetesAssociation, American Heart Association, American Association ofDiabetes Educators, European Society of Endocrinology, and the Societyof Hospital Medicine (Korytkowski M, McDonnell M E, Umpierrez G E,Zonszein J, J Clin Endocrinol Metab. (2012), January; 97(1): 27A-8A;Umpierrez G E, Hellman R, Korytkowski M T, Kosiborod M, Maynard G A,Montori V M, et al. J Clin Endocrinol Metab. (2012) January; 97(1):16-38).

However, while insulin therapy appears to be the only acceptabletreatment for stress hyperglycemia, intensive insulin treatment totarget optimal blood glucose levels may result in hypoglycemia, thusreversing the beneficial effects of insulin therapy. Such insulintherapy may actually increase the risk of mortality in this patientpopulation. Thus, a medical need exists for new parenteral agents, suchas a monoclonal antibody that blocks glucagon action, to improve thetreatment and outcome of patients with stress hyperglycemia.

Accordingly, it is an object of the present invention to treat stresshyperglycemia with an anti-GCGR antibody of the invention, either alone,or in conjunction with at least one other glucose lowering agent. It isan object of the invention to use an anti-GCGR antibody in combinationwith insulin or potentially as monotherapy to treat stress hyperglycemiain both the critically ill and not critically ill hospitalized patient.It is an object of the invention to provide beneficial effects in thispatient population through use of an anti-GCGR antibody of theinvention. Such beneficial effects include the following: reducedglucagon-dependent metabolic perturbations such as hyperglycemia andincreased catabolism; reduced risk of hypoglycemia; decreased morbidityand mortality (e.g., organ failure and infection); and/or decreasedhospital resource utilization (length of stay, nursing care, time inICU).

It is also an object of the invention to prevent the onset ofhyperglycemia in a patient at risk for developing stress hyperglycemiaby prophylactic administration of an antibody of the invention, eitheras stand alone therapy, or in conjunction with at least one otherglucose lowering agent, for example, insulin. A patient “at risk fordeveloping stress hyperglycemia” is defined herein as an individual whohas been exposed to, or is currently being exposed to, or may be exposedto, certain stressors that may result in a rise in blood glucose levelsabove normal. These stressors may include, but are not limited to,anesthesia, surgery, trauma, an underlying medical illness, or any otherrisk factor selected from the group consisting of type 1 or type 2diabetes; infusion of catecholamine pressors; glucocorticoid therapy;obesity; aging; excessive dextrose administration; parenteral nutrition,enteral nutrition, pancreatitis; sepsis; stroke; traumatic head injury;hypothermia; hypoxemia; uremia; cirrhosis; anesthesia; pre-operative orpost-operative hospital stays (pen-operative hyperglycemia); admissionto an emergency room, a trauma center, or an intensive care unit;prolonged hospital stays; an infection; or a worsening chronic illness.

The administration of insulin has in certain instances been associatedwith a sudden drop in blood glucose (hypoglycemia) and puts the patientat risk for greater morbidity and mortality. Certain studies reported onherein have demonstrated an insulin sparing effect through use ofcertain antibodies of the invention in conjunction with insulin.Accordingly, based on these studies, it is a further object of theinvention to be able to lower the amount of insulin administered topatients if the anti-GCGR antibodies of the invention are administeredconcurrently with insulin. Such concomitant administration of both theanti-GCGR antibody plus lower amounts of insulin can result in thedesired effect without the risk of increased morbidity and/or mortality.

Antigen-Binding Fragments of Antibodies

Unless specifically indicated otherwise, the term “antibody,” as usedherein, shall be understood to encompass antibody molecules comprisingtwo immunoglobulin heavy chains and two immunoglobulin light chains(i.e., “full antibody molecules”) as well as antigen-binding fragmentsthereof. The terms “antigen-binding portion” of an antibody,“antigen-binding fragment” of an antibody, and the like, as used herein,include any naturally occurring, enzymatically obtainable, synthetic, orgenetically engineered polypeptide or glycoprotein that specificallybinds an antigen to form a complex. The terms “antigen-binding portion”of an antibody, or “antibody fragment”, as used herein, refers to one ormore fragments of an antibody that retain the ability to specificallybind to hGCGR. An antibody fragment may include a Fab fragment, aF(ab′)₂ fragment, a Fv fragment, a dAb fragment, a fragment containing aCDR, or an isolated CDR. Antigen-binding fragments of an antibody may bederived, e.g., from full antibody molecules using any suitable standardtechniques such as proteolytic digestion or recombinant geneticengineering techniques involving the manipulation and expression of DNAencoding antibody variable and (optionally) constant domains. Such DNAis known and/or is readily available from, e.g., commercial sources, DNAlibraries (including, e.g., phage-antibody libraries), or can besynthesized. The DNA may be sequenced and manipulated chemically or byusing molecular biology techniques, for example, to arrange one or morevariable and/or constant domains into a suitable configuration, or tointroduce codons, create cysteine residues, modify, add or delete aminoacids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDR,which is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)—V_(H), V_(H)—V_(L) orV_(L)—V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)—C_(H)1; (ii)V_(H)—C_(H)2; (iii) V_(H)—C_(H)3; (iv) V_(H)—C_(H)1—C_(H)2; (v)V_(H)—C_(H)1—C_(H)2—C_(H)3, (vi) V_(H)—C_(H)2—C_(H)3; (vii) V_(H)—C_(L);(viii) V_(L)—C_(H)1; (ix) V_(L)—C_(H)2; (x) V_(L)—C_(H)3; (xi)V_(L)—C_(H)1—C_(H)2; (xii) V_(L)—C_(H)1—C_(H)2—C_(H)3; (xiii)V_(L)—C_(H)2—C_(H)3; and (xiv) V_(L)—C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemono-specific or multi-specific (e.g., bi-specific). A multi-specificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multi-specific antibody format, including theexemplary bi-specific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

Preparation of Human Antibodies

Methods for generating human antibodies in transgenic mice are known inthe art. Any such known methods can be used in the context of thepresent invention to make human antibodies that specifically bind tohuman GCGR.

Using VELOCIMMUNE® technology (see, for example, U.S. Pat. No.6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other knownmethod for generating monoclonal antibodies, high affinity chimericantibodies to GCGR are initially isolated having a human variable regionand a mouse constant region. The VELOCIMMUNE® technology involvesgeneration of a transgenic mouse having a genome comprising human heavyand light chain variable regions operably linked to endogenous mouseconstant region loci such that the mouse produces an antibody comprisinga human variable region and a mouse constant region in response toantigenic stimulation. The DNA encoding the variable regions of theheavy and light chains of the antibody are isolated and operably linkedto DNA encoding the human heavy and light chain constant regions. TheDNA is then expressed in a cell capable of expressing the fully humanantibody.

Generally, a VELOCIMMUNE® mouse is challenged with the antigen ofinterest, and lymphatic cells (such as B-cells) are recovered from themice that express antibodies. The lymphatic cells may be fused with amyeloma cell line to prepare immortal hybridoma cell lines, and suchhybridoma cell lines are screened and selected to identify hybridomacell lines that produce antibodies specific to the antigen of interest.DNA encoding the variable regions of the heavy chain and light chain maybe isolated and linked to desirable isotypic constant regions of theheavy chain and light chain. Such an antibody protein may be produced ina cell, such as a CHO cell. Alternatively, DNA encoding theantigen-specific chimeric antibodies or the variable domains of thelight and heavy chains may be isolated directly from antigen-specificlymphocytes.

Initially, high affinity chimeric antibodies are isolated having a humanvariable region and a mouse constant region. As in the experimentalsection below, the antibodies are characterized and selected fordesirable characteristics, including affinity, selectivity, epitope,etc. The mouse constant regions are replaced with a desired humanconstant region to generate the fully human antibody of the invention,for example wild-type or modified IgG1 or IgG4. While the constantregion selected may vary according to specific use, high affinityantigen-binding and target specificity characteristics reside in thevariable region.

In general, the antibodies of the instant invention possess very highaffinities, typically possessing K_(D) of from about 10⁻¹² through about10⁻⁹ M, when measured by binding to antigen either immobilized on solidphase or in solution phase. The mouse constant regions are replaced withdesired human constant regions to generate the fully human antibodies ofthe invention. While the constant region selected may vary according tospecific use, high affinity antigen-binding and target specificitycharacteristics reside in the variable region.

Bioequivalents

The anti-GCGR antibodies and antibody fragments of the present inventionencompass proteins having amino acid sequences that vary from those ofthe described antibodies, but that retain the ability to bind humanGCGR. Such variant antibodies and antibody fragments comprise one ormore additions, deletions, or substitutions of amino acids when comparedto parent sequence, but exhibit biological activity that is essentiallyequivalent to that of the described antibodies. Likewise, the anti-GCGRantibody-encoding DNA sequences of the present invention encompasssequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to the disclosed sequence,but that encode an anti-GCGR antibody or antibody fragment that isessentially bioequivalent to an anti-GCGR antibody or antibody fragmentof the invention.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and/or in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-GCGR antibodies of the invention may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation. In other contexts,bioequivalent antibodies may include anti-GCGR antibody variantscomprising amino acid changes, which modify the glycosylationcharacteristics of the antibodies, e.g., mutations which eliminate orremove glycosylation.

Biological Characteristics of the Antibodies

In general, the antibodies of the present invention may function bybinding to at least one of the extracellular regions of hGCGR. Incertain embodiments, the antibodies of the present invention may bind toan epitope located in at least the N-terminal region, or to an epitopelocated in at least one of the extracellular (EC) loops of hGCGR.

In certain embodiments, the antibodies of the present invention mayfunction by blocking or inhibiting GCGR activity by binding to theextracellular N-terminal region, the amino acid sequence of which isshown in SEQ ID NO: 159, and which is encoded by the nucleic acidsequence shown in SEQ ID NO: 158.

In certain embodiments, the antibodies of the present invention mayfunction by blocking or inhibiting GCGR activity by binding to at leastone of the EC loops or loop segments within the whole receptor. In oneembodiment, the antibodies of the invention may bind to an epitopelocated in EC1, which is located between about amino acid residue 194 toabout amino acid residue 226 of SEQ ID NO: 153. Alternatively, oradditionally, the antibodies of the invention may bind to an epitopefound in EC2, which is located between about amino acid residue 285 toabout amino acid residue 302 of SEQ ID NO: 153. Alternatively, oradditionally, the antibodies of the invention may bind to an epitopefound in EC3, which is located between about amino acid residue 369 toabout amino acid residue 384 of SEQ ID NO: 153.

In certain embodiments, the antibodies of the present invention may bebi-specific antibodies. The bi-specific antibodies of the invention maybind one epitope in EC1 and may also bind one epitope in a region ofhGCGR other than EC1. In certain embodiments, the bi-specific antibodiesof the invention may bind one epitope in EC1 and may also bind oneepitope in EC2 or EC3, or in the N-terminal region, or in any otherregion within EC1, EC2, or EC3 of hGCGR, or any combination thereof. Incertain embodiments, the bi-specific antibodies of the invention maybind to two different sites within the same extracellular region.

More specifically, the anti-GCGR antibodies of the invention may exhibitone or more of the following characteristics: (1) ability to bind to ahuman GCGR or a fragment thereof and to a non-human (e.g., mouse,monkey, rat, rabbit, dog, pig, etc.) GCGR or fragment thereof; (2)ability to bind to a human GCGR or fragment thereof, but not to anon-human (e.g., mouse, monkey, rat, rabbit, dog, pig, etc.) GCGR orfragment thereof; (3) ability to bind to a human GCGR or fragmentthereof and to a non-human primate (e.g. monkey) GCGR or fragmentthereof, but not to a mouse, rat, rabbit, dog or pig GCGR or GCGRfragment; (4) ability to bind to a human GCGR or fragment thereof and toa non-human primate (e.g. monkey) GCGR or a fragment thereof, and to amouse GCGR or a fragment thereof, but not to a rat GCGR; (5) ability tobind to a human GCGR or fragment thereof and to a non-human primate(e.g. monkey) GCGR or a fragment thereof, and to a rat GCGR or afragment thereof, but not to a mouse GCGR; 6) blocks glucagon binding toGCGR; 7) blocks glucagon induced cAMP production; 8) demonstrates theability to lower blood glucose levels in humans suffering from diabetesand in animal models of diabetes; 9) may or may not lower triglyceridelevels to the levels observed in normal mammals; or 10) does notadversely affect plasma lipid levels.

Certain anti-GCGR antibodies of the present invention are able toinhibit or attenuate GCGR activity in an in vitro or in vivo assay. Theability of the antibodies of the invention to bind to and inhibitbinding of glucagon to GCGR may be measured using any standard methodknown to those skilled in the art, including binding assays, reporterbioassays, such as a luciferase reporter assay.

Non-limiting, exemplary in vitro assays for measuring GCGR activity areillustrated in Examples 4 and 5, herein. In Example 4, the bindingaffinities and kinetic constants of human anti-hGCGR antibodies weredetermined by surface plasmon resonance and the measurements wereconducted on a T100 Biacore instrument. In Example 5, a bioassay wasdeveloped in HEK293 cell lines expressing full length human, monkey andmouse GCGR along with a luciferase reporter in order to detectactivation through Gαs, and subsequent elevation of cAMP levels andtranscriptional activation. Examples 6, 7, 8, 9 and 10 demonstrate thein vivo effects of the antibodies on lowering of blood glucose levels,blood ketone levels, and on weight loss, in various animal models.

The present invention also includes anti-GCGR antibodies and antigenbinding fragments thereof which bind to at least one biologically activefragment of any of the following proteins, or peptides: SEQ ID NO: 153(full length hGCGR), residue numbers 27-144 of SEQ ID NO: 153(N-terminal domain of hGCGR); residues 194-226 of SEQ ID NO: 153;residues 285-305 of SEQ ID NO: 153; residues 369-384 of SEQ ID NO: 153.Any of the GCGR peptides described herein, or fragments thereof, may beused to generate anti-GCGR antibodies.

The peptides may be modified to include addition or substitution ofcertain residues for tagging or for purposes of conjugation to carriermolecules, such as, KLH. For example, a cysteine may be added at eitherthe N terminal or C terminal end of a peptide, or a linker sequence maybe added to prepare the peptide for conjugation to, for example, KLH forimmunization. The antibodies specific for GCGR may contain no additionallabels or moieties, or they may contain an N-terminal or C-terminallabel or moiety. In one embodiment, the label or moiety is biotin. In abinding assay, the location of a label (if any) may determine theorientation of the peptide relative to the surface upon which thepeptide is bound. For example, if a surface is coated with avidin, apeptide containing an N-terminal biotin will be oriented such that theC-terminal portion of the peptide will be distal to the surface.

In one embodiment, the invention provides a fully human monoclonalantibody or antigen-binding fragment thereof that specifically bindshGCGR and neutralizes hGCGR activity, wherein the antibody or fragmentthereof exhibits one or more of the following characteristics: (i)comprises a HCVR having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126,130, and 146; (ii) comprises a LCVR having an amino acid sequenceselected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 68, 78,88, 98, 108, 118, 128, 138, and 148; (iii) comprises any one or more ofthe heavy chain CDR1 sequences selected from the group consisting of 4,20, 36, 52, 72, 92, 112 and 132; any one or more of the heavy chain CDR2sequences selected from the group consisting of 6, 22, 38, 54, 74, 94,114 and 134; any one or more of the heavy chain CDR3 sequences selectedfrom the group consisting of 8, 24, 40, 56, 76, 96, 116 and 136; any oneor more of the light chain CDR1 sequences selected from the groupconsisting of 12, 28, 44, 60, 80, 100, 120 and 140; any one or more ofthe light chain CDR2 sequences selected from the group consisting of 14,30, 46, 62, 82, 102, 122 and 142; any one or more of the light chainCDR3 sequences selected from the group consisting of 16, 32, 48, 64, 84,104, 124 and 144; and combinations thereof; (iv) demonstrates bindingspecificity for any one or more of the following: the N-terminal regionof GCGR comprising amino acid residues 27-144 of SEQ ID NO: 153, or forany one or more of the extracellular loops of GCGR, including, forexample, EC1, EC2, or EC3, wherein EC1 comprises amino acid residuesranging from about residue 194 to about residue 226 of SEQ ID NO: 153,and wherein EC2 comprises amino acid residues ranging from about residue285 to about residue 305 of SEQ ID NO: 153; and wherein EC3 comprisesamino acid residues ranging from about residue 369 to about residue 384of SEQ ID NO: 153; (v) binds any one or more of human, monkey, mouse orrat GCGR; (vi) blocks binding of glucagon to GCGR; vi) blocks glucagoninduced cAMP production; vii) demonstrates the ability to lower bloodglucose levels or blood ketone levels in humans suffering from diabetesor in animal models of diabetes; viii) may or may not lower triglyceridelevels to the levels observed in normal mammals; or ix) does notadversely affect plasma lipid levels.

Epitope Mapping and Related Technologies

The term “epitope,” as used herein, refers to an antigenic determinantthat interacts with a specific antigen binding site in the variableregion of an antibody molecule known as a paratope. A single antigen mayhave more than one epitope. Thus, different antibodies may bind todifferent areas on an antigen and may have different biological effects.Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain. Incertain circumstance, an epitope may include moieties of saccharides,phosphoryl groups, or sulfonyl groups on the antigen. An epitopetypically includes at least 3, and more usually, at least 5 or 8-10amino acids in a unique spatial conformation.

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antibody “interacts with one or more aminoacids” within a polypeptide or protein, or binds to a particular epitopewithin a polypeptide or protein. Exemplary techniques include, forexample, routine cross-blocking assays, such as that described inAntibodies, Harlow and Lane (Cold Spring Harbor Press, Cold SpringHarb., NY). Other methods include alanine scanning mutational analysis,peptide blot analysis (Reineke (2004) Methods Mol Biol 248:443-63),peptide cleavage analysis crystallographic studies and NMR analysis. Inaddition, methods such as epitope excision, epitope extraction andchemical modification of antigens can be employed (Tomer (2000) ProteinScience 9: 487-496). Another method that can be used to identify theamino acids within a polypeptide with which an antibody interacts ishydrogen/deuterium exchange detected by mass spectrometry. In generalterms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding theantibody to the deuterium-labeled protein. Next, the protein/antibodycomplex is transferred to water and exchangeable protons within aminoacids that are protected by the antibody complex undergodeuterium-to-hydrogen back-exchange at a slower rate than exchangeableprotons within amino acids that are not part of the interface. As aresult, amino acids that form part of the protein/antibody interface mayretain deuterium and therefore exhibit relatively higher mass comparedto amino acids not included in the interface. After dissociation of theantibody, the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residueswhich correspond to the specific amino acids with which the antibodyinteracts. See, e.g., Ehring (1999) Analytical Biochemistry267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256 A-265A. X-raycrystallography of the antigen/antibody complex may also be used forepitope mapping purposes.

Modification-Assisted Profiling (MAP), also known as AntigenStructure-based Antibody Profiling (ASAP) is a method that categorizeslarge numbers of monoclonal antibodies (mAbs) directed against the sameantigen according to the similarities of the binding profile of eachantibody to chemically or enzymatically modified antigen surfaces (US2004/0101920, herein specifically incorporated by reference in itsentirety). Each category may reflect a unique epitope either distinctlydifferent from or partially overlapping with epitope represented byanother category. This technology allows rapid filtering of geneticallyidentical antibodies, such that characterization can be focused ongenetically distinct antibodies. When applied to hybridoma screening,MAP may facilitate identification of rare hybridoma clones that producemAbs having the desired characteristics. MAP may be used to sort theanti-GCGR antibodies of the invention into groups of antibodies bindingdifferent epitopes.

In certain embodiments, the anti-GCGR antibody or antigen-bindingfragment of an antibody binds an epitope within at least one of theextracellular regions of GCGR, or to a fragment thereof, wherein theextracellular region is the N-terminal domain, or one of the EC loops,including EC1, EC2, or EC3, as described previously.

In one embodiment, the antibody binds an epitope within the N-terminalregion of GCGR, or a fragment thereof, comprising an amino acid sequenceranging from about amino acid residue 27-144 of SEQ ID NO: 153. In oneembodiment, the antibody binds an epitope within EC1, or a fragmentthereof, comprising an amino acid sequence ranging from about amino acidresidue 194-226 of SEQ ID NO: 153. In one embodiment, the antibody bindsan epitope within EC2, or a fragment thereof, comprising an amino acidsequence ranging from amino acid residue 285-305 of SEQ ID NO: 153. Inone embodiment, the antibody binds an epitope within EC3, or a fragmentthereof, comprising an amino acid sequence ranging from about amino acidresidue 369-384 of SEQ ID NO: 153.

In certain embodiments, the antibody or antibody fragment binds anepitope which includes more than one of the enumerated epitopes of GCGRwithin the N-terminal domain, or within EC1, EC2, or EC3, and/or withintwo or three different extracellular regions (for example, epitopeswithin the N-terminal region, EC1, EC2 and EC3 loops, or within EC1,EC2, and EC3, or within the N-terminal region, EC2 and EC3 loops, orwithin the N-terminal region, EC1 and EC3 loops.

In certain embodiments, the antibody is a bi-specific antibody thatbinds one epitope within one extracellular region of GCGR and anotherepitope within a different extracellular region of GCGR, including theN-terminal domain, or EC1, EC2, or EC3.

In one embodiment, the antibody is a bi-specific antibody that binds oneepitope in the N-terminal region of hGCGR and another epitope in EC1 ofhGCGR. In one embodiment, the antibody is a bi-specific antibody thatbinds one epitope in the N-terminal region of hGCGR and another epitopein EC1 of hGCGR. In one embodiment, the antibody is a bi-specificantibody that binds one epitope in the N-terminal region of hGCGR andanother epitope in EC2 of hGCGR. In one embodiment, the antibody is abi-specific antibody that binds one epitope in the N-terminal region ofhGCGR and another epitope in EC3 of hGCGR. In one embodiment, theantibody is a bi-specific antibody that binds one epitope in EC1 ofhGCGR and another epitope in EC2 of hGCGR. In one embodiment, theantibody is a bi-specific antibody that binds one epitope in EC1 ofhGCGR and another epitope in EC3 of hGCGR. In one embodiment, theantibody is a bi-specific antibody that binds one epitope in EC2 ofhGCGR and another epitope in EC3 of hGCGR.

In one embodiment, the antibody is a bi-specific antibody that binds oneepitope in the N terminal domain of hGCGR, wherein the one epitoperanges from about residue 27 to about residue 144 of SEQ ID NO: 153 anda second epitope in EC1 of hGCGR, wherein the second epitope ranges fromabout residue number 194 to about residue number 226 of SEQ ID NO: 153.In one embodiment, the antibody is a bi-specific antibody that binds oneepitope in the N terminal domain of hGCGR within the residues notedabove, and a second epitope in EC2 of hGCGR, wherein the second epitoperanges from about residue number 285 to about residue number 305 of SEQID NO:153. In one embodiment, the antibody is a bi-specific antibodythat binds one epitope in the N terminal domain of hGCGR within theresidues noted above, and a second epitope in EC3 of hGCGR, wherein thesecond epitope ranges from about residue number 369 to about residuenumber 384 of SEQ ID NO: 153.

In one embodiment, the antibody is a bi-specific antibody that binds oneepitope in EC1 of hGCGR from about residue 194 to about residue 226 ofSEQ ID NO: 153 and a second epitope in EC2 of GCGR from about residue285 to about residue 305 of SEQ ID NO: 153. In one embodiment, theantibody is a bi-specific antibody that binds one epitope in EC1 fromabout residue 194 to about residue 226 of SEQ ID NO: 153 and a secondepitope in EC3 of GCGR from about residue 369 to about residue 384 ofSEQ ID NO:153. In one embodiment, the antibody is a bi-specific antibodythat binds one epitope in EC2 from about residue 285 to about residue305 of SEQ ID NO:153 and a second epitope in EC3 of GCGR from aboutresidue 369 to about residue 384 of SEQ ID NO:153.

The present invention includes anti-GCGR antibodies that bind to thesame epitope as any of the specific exemplary antibodies describedherein (e.g., H4H1345N, H4H1617N, H4H1765N, H4H1321B and H4H1321P,H4H1327B and H4H1327P, H4H1328B and H4H1328P, H4H1331B and H4H1331P,H4H1339B and H4H1339P). Likewise, the present invention also includesanti-GCGR antibodies that compete for binding to GCGR or a GCGR fragmentwith any of the specific exemplary antibodies described herein.

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-GCGR antibody byusing routine methods known in the art. For example, to determine if atest antibody binds to the same epitope as a reference anti-GCGRantibody of the invention, the reference antibody is allowed to bind toa GCGR protein or peptide under saturating conditions. Next, the abilityof a test antibody to bind to the GCGR molecule is assessed. If the testantibody is able to bind to GCGR following saturation binding with thereference anti-GCGR antibody, it can be concluded that the test antibodybinds to a different epitope than the reference anti-GCGR antibody. Onthe other hand, if the test antibody is not able to bind to the GCGRmolecule following saturation binding with the reference anti-GCGRantibody, then the test antibody may bind to the same epitope as theepitope bound by the reference anti-GCGR antibody of the invention.

To determine if an antibody competes for binding with a referenceanti-GCGR antibody, the above-described binding methodology is performedin two orientations: In a first orientation, the reference antibody isallowed to bind to a GCGR molecule under saturating conditions followedby assessment of binding of the test antibody to the GCGR molecule. In asecond orientation, the test antibody is allowed to bind to a GCGRmolecule under saturating conditions followed by assessment of bindingof the reference antibody to the GCGR molecule. If, in bothorientations, only the first (saturating) antibody is capable of bindingto the GCGR molecule, then it is concluded that the test antibody andthe reference antibody compete for binding to GCGR. As will beappreciated by a person of ordinary skill in the art, an antibody thatcompetes for binding with a reference antibody may not necessarily bindto the identical epitope as the reference antibody, but may stericallyblock binding of the reference antibody by binding an overlapping oradjacent epitope.

Two antibodies bind to the same or overlapping epitope if eachcompetitively inhibits (blocks) binding of the other to the antigen.That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibitsbinding of the other by at least 50% but preferably 75%, 90% or even 99%as measured in a competitive binding assay (see, e.g., Junghans et al.,Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have thesame epitope if essentially all amino acid mutations in the antigen thatreduce or eliminate binding of one antibody reduce or eliminate bindingof the other. Two antibodies have overlapping epitopes if some aminoacid mutations that reduce or eliminate binding of one antibody reduceor eliminate binding of the other.

Additional routine experimentation (e.g., peptide mutation and bindinganalyses) can then be carried out to confirm whether the observed lackof binding of the test antibody is in fact due to binding to the sameepitope as the reference antibody or if steric blocking (or anotherphenomenon) is responsible for the lack of observed binding. Experimentsof this sort can be performed using ELISA, RIA, surface plasmonresonance, flow cytometry or any other quantitative or qualitativeantibody-binding assay available in the art.

Species Selectivity and Species Cross-Reactivity

According to certain embodiments of the invention, the anti-GCGRantibodies bind to human GCGR but not to GCGR from other species.Alternatively, the anti-GCGR antibodies of the invention, in certainembodiments, bind to human GCGR and to GCGR from one or more non-humanspecies. For example, the anti-GCGR antibodies of the invention may bindto human GCGR and may bind or not bind, as the case may be, to one ormore of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit,goat, sheep, cow, horse, camel, cynomolgus, marmoset, rhesus orchimpanzee GCGR.

Immunoconjugates

The invention encompasses a human anti-GCGR monoclonal antibodyconjugated to a therapeutic moiety (“immunoconjugate”), such as an agentthat is capable of reducing blood glucose levels, or a radioisotope, ora chemotherapeutic agent. The type of therapeutic moiety that may beconjugated to the anti-GCGR antibody will take into account thecondition to be treated and the desired therapeutic effect to beachieved. For example, for treating diabetes, or any other conditionwhereby it is desirable to lower blood glucose, and/or to maintainnormal blood glucose levels, an agent such as biguanide (e.g.metformin), a sulfonylurea (e.g. glyburide, glipizide), a PPAR gammaagonist (e.g. pioglitazone, rosiglitazone); an alpha glucosidaseinhibitor (e.g. acarbose, voglibose), an inhibitor of advanced glycationendproduct formation (e.g. aminoguanidine), or a second GCGR inhibitormay be conjugated to the GCGR antibody. Alternatively, if the desiredtherapeutic effect is to treat the sequelae or symptoms associated withdiabetes, or any other condition resulting from high, or uncontrolledblood glucose levels, it may be advantageous to conjugate an agentappropriate to treat the sequelae or symptoms of the condition Examplesof suitable agents for forming immunoconjugates are known in the art,see for example, WO 05/103081.

Multi-Specific Antibodies

The antibodies of the present invention may be mono-specific,bi-specific, or multi-specific. Multi-specific antibodies may bespecific for different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244. The anti-GCGR antibodies of the presentinvention can be linked to or co-expressed with another functionalmolecule, e.g., another peptide or protein. For example, an antibody orfragment thereof can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody or antibody fragmentto produce a bi-specific or a multi-specific antibody with a secondbinding specificity. For example, the present invention includesbi-specific antibodies wherein one arm of an immunoglobulin is specificfor human GCGR or a fragment thereof, and the other arm of theimmunoglobulin is specific for a second therapeutic target or isconjugated to a therapeutic moiety. In certain embodiments of theinvention, one arm of an immunoglobulin is specific for an epitope onthe N-terminal domain of hGCGR or a fragment thereof, and the other armof the immunoglobulin is specific for an epitope on one of the EC loopsof hGCGR, or a fragment thereof. In certain embodiments, one arm of animmunoglobulin is specific for one EC loop, or a fragment thereof, andthe second arm is specific for a second EC loop, or a fragment thereof.In certain embodiments, one arm of an immunoglobulin is specific for oneepitope on one EC loop of hGCGR and the other arm is specific for asecond epitope on the same EC loop of hGCGR.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bi-specific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise a Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies. Variations on the bi-specificantibody format described above are contemplated within the scope of thepresent invention.

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising the anti-GCGRantibodies or antigen-binding fragments thereof of the presentinvention. The administration of therapeutic compositions in accordancewith the invention will be administered with suitable carriers,excipients, and other agents that are incorporated into formulations toprovide improved transfer, delivery, tolerance, and the like. Amultitude of appropriate formulations can be found in the formularyknown to all pharmaceutical chemists: Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa. These formulationsinclude, for example, powders, pastes, ointments, jellies, waxes, oils,lipids, lipid (cationic or anionic) containing vesicles (such asLIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-waterand water-in-oil emulsions, emulsions carbowax (polyethylene glycols ofvarious molecular weights), semi-solid gels, and semi-solid mixturescontaining carbowax. See also Powell et al. “Compendium of excipientsfor parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

The dose of antibody may vary depending upon the age and the size of asubject to be administered, target disease, conditions, route ofadministration, and the like. When the antibody of the present inventionis used for lowering blood glucose levels associated with GCGR activityin various conditions and diseases, such as diabetes, in an adultpatient, it is advantageous to intravenously administer the antibody ofthe present invention normally at a single dose of about 0.01 to about30 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03to about 5, or about 0.05 to about 3 mg/kg body weight. Depending on theseverity of the condition, the frequency and the duration of thetreatment can be adjusted. In certain embodiments, the antibody orantigen-binding fragment thereof of the invention can be administered asan initial dose of at least about 0.1 mg to about 800 mg, about 1 toabout 500 mg, about 5 to about 300 mg, or about 10 to about 200 mg, toabout 100 mg, or to about 50 mg. In certain embodiments, the initialdose may be followed by administration of a second or a plurality ofsubsequent doses of the antibody or antigen-binding fragment thereof inan amount that can be approximately the same or less than that of theinitial dose, wherein the subsequent doses are separated by at least 1day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; atleast 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; atleast 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks;or at least 14 weeks.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, transdermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural and oral routes. The composition may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local.

The pharmaceutical composition can be also delivered in a vesicle, inparticular a liposome (see, for example, Langer (1990) Science249:1527-1533).

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used.In another embodiment, polymeric materials can be used. In yet anotherembodiment, a controlled release system can be placed in proximity ofthe composition's target, thus requiring only a fraction of the systemicdose.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but certainlyare not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™,OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis,Frankfurt, Germany), to name only a few. Examples of disposable pendelivery devices having applications in subcutaneous delivery of apharmaceutical composition of the present invention include, butcertainly are not limited to the SOLOSTAR™ pen (sanofi-aventis), theFLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™Autoinjector (Amgen, Thousands Oaks, Calif.), the PENLET™ (Haselmeier,Stuttgart, Germany), the EPIPEN® (Dey, L. P.) and the HUMIRA™ Pen(Abbott Labs, Abbott Park, Ill.), to name only a few.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antibodies

Due to their interaction with the glucagon receptor, the presentantibodies are useful for lowering blood glucose levels and also for thetreatment of a wide range of conditions and disorders in which blockingthe interaction of glucagon with its receptor is beneficial. Thesedisorders and conditions may be selected from any glucagon relatedmetabolic disorder, which involves glucagon receptor signaling thatresults in the pathophysiology of the disorder, or in the homeostaticresponse to the disorder. Thus, the antibodies may find use for exampleto prevent, treat, or alleviate, diseases or conditions or associatedsymptoms or sequelae, of the endocrine system, the central nervoussystem, the peripheral nervous system, the cardiovascular system, thepulmonary system, and the gastrointestinal system, while reducing and oreliminating one or more of the unwanted side effects associated with thecurrent treatments. Glucagon related metabolic disorders include, butare not limited to, type 1 and type 2 diabetes, diabetic ketoacidosis,hyperglycemia, hyperglycemic hyperosmolar syndrome, perioperativehyperglycemia, hyperglycemia in the intensive care unit patient,hyperinsulinemia, postprandial hyperglycemia, impaired fasting glucose(IFG), metabolic syndrome, hyper-/hypokalemia, poor LDL/HDL ratio,eating disorders, weight gain, obesity as a consequence of diabetes,pediatric diabetes, gestational diabetes, diabetic late complications,micro-/macroalbuminuria, nephropathy, retinopathy, neuropathy, diabeticfoot ulcers, wound healing, impaired glucose tolerance (IGT), insulinresistance syndromes, syndrome X, glucagonomas, gastrointestinaldisorders, obesity, diabetes as a consequence of obesity, etc. Thepresent invention further provides; a method of treating conditionsresulting from excessive glucagon in a mammal; a method of inhibitingthe glucagon receptor in a mammal; a method of inhibiting a glucagonreceptor mediated cellular response in a mammal, or a method of reducingthe glycemic level in a mammal comprising administering to a mammal inneed of such treatment a glucagon receptor-inhibiting amount of ananti-GCGR antibody or a biologically active fragment thereof.

The present antibodies are effective in lowering blood glucose, both inthe fasting and the postprandial stage. In certain embodiments of theinvention, the present antibodies are used for the preparation of apharmaceutical composition for the treatment of type 2 diabetes. In yeta further embodiment of the invention the present antibodies are usedfor the preparation of a pharmaceutical composition for the delaying orprevention of the progression from impaired glucose tolerance to type 2diabetes. In yet another embodiment of the invention the presentantibodies are used for the preparation of a pharmaceutical compositionfor the delaying or prevention of the progression from non-insulinrequiring diabetes to insulin requiring diabetes. In a furtherembodiment of the invention the present antibodies are used for thepreparation of a pharmaceutical composition for the treatment of type 1diabetes. Such treatment is normally accompanied by insulin therapy.

Combination Therapies

Combination therapies may include an anti-hGCGR antibody of theinvention and any additional therapeutic agent that may beadvantageously combined with an antibody of the invention, or with abiologically active fragment of an antibody of the invention.

For example, a second therapeutic agent may be employed to aid infurther lowering of glucose levels, or to reduce at least one symptom ina patient suffering from a disease or condition characterized by highblood glucose levels, such as diabetes mellitus. Such a second agent maybe selected from, for example, a glucagon antagonist or another GCGRantagonist (e.g. an anti-glucagon or anti-GCGR antibody or smallmolecule inhibitor of glucagon or GCGR), or may include othertherapeutic moieties useful for treating diabetes, or other diseases orconditions associated with, or resulting from elevated blood glucoselevels, or impaired glucose metabolism, or agents useful for treatingany long term complications associated with elevated and/or uncontrolledblood glucose levels. These agents include biguanides, which decreaseglucose production in the liver and increase sensitivity to insulin(e.g. metformin), or sulfonylureas, which stimulate insulin production(e.g. glyburide, glipizide). Additional treatments directed atmaintaining glucose homeostasis including PPAR gamma agonists, which actas insulin sensitizers (e.g. pioglitazone, rosiglitazone); and alphaglucosidase inhibitors, which slow starch absorption and glucoseproduction (e.g. acarbose, voglibose). Additional treatments includeinjectable treatments such as Exenatide® (glucagon-like peptide 1), andSymlin® (pramlintide).

In certain other embodiments, the composition may include a second agentselected from the group consisting of non-sulfonylurea secretagogues,insulin, insulin analogs, exendin-4 polypeptides, beta 3 adrenoceptoragonists, PPAR agonists, dipeptidyl peptidase IV inhibitors, statins andstatin-containing combinations, cholesterol absorption inhibitors,LDL-cholesterol antagonists, cholesteryl ester transfer proteinantagonists, endothelin receptor antagonists, growth hormoneantagonists, insulin sensitizers, amylin mimetics or agonists,cannabinoid receptor antagonists, glucagon-like peptide-1 agonists,melanocortins, melanin-concentrating hormone receptor agonists, SNRIs,and protein tyrosine phosphatase inhibitors.

In certain other embodiments, combination therapy may includeadministration of a second agent to counteract any potential sideeffect(s) resulting from administration of an antibody of the invention,if such side effect(s) occur. For example, in the event that any of theanti-GCGR antibodies increases lipid or cholesterol levels, it may bebeneficial to administer a second agent to lower lipid or cholesterollevels, using an agent such as a HMG-CoA reductase inhibitor (forexample, a statin such as atorvastatin, (LIPITOR®), fluvastatin(LESCOL®), lovastatin (MEVACOR®), pitavastatin (LIVALO®), pravastatin(PRAVACHOL®), rosuvastatin (CRESTOR®) and simvastatin (ZOCOR®).Alternatively, the antibodies of the invention may be combined with anagent such as VYTORIN®, which is a preparation of a statin and anotheragent—such as ezetimibe/simvastatin.

In certain embodiments, it may be beneficial to administer theantibodies of the invention in combination with any one or more of thefollowing: (1) niacin, which increases lipoprotein catabolism; (2)fibrates or amphipathic carboxylic acids, which reduce low-densitylipoprotein (LDL) level, improve high-density lipoprotein (HDL) and TGlevels, and reduce the number of non-fatal heart attacks; and (3)activators of the LXR transcription factor that plays a role incholesterol elimination such as 22-hydroxycholesterol, or a statin witha bile resin (e.g., cholestyramine, colestipol, colesevelam), a fixedcombination of niacin plus a statin (e.g., niacin with lovastatin); orwith other lipid lowering agents such as omega-3-fatty acid ethyl esters(for example, omacor).

Furthermore, the second therapeutic agent can be one or more otherinhibitors of glucagon or GCGR, as well as inhibitors of othermolecules, such as angiopoietin-like protein 3 (ANGPTL3),angiopoietin-like protein 4 (ANGPTL4), angiopoietin-like protein 5(ANGPTL5), angiopoietin-like protein 6 (ANGPTL6), which are involved inlipid metabolism, in particular, cholesterol and/or triglyceridehomeostasis. Inhibitors of these molecules include small molecules andantibodies that specifically bind to these molecules and block theiractivity.

In certain embodiments, it may be beneficial to administer theantibodies of the invention in combination with an antibody that acts tolower lipid or cholesterol levels, such as, but not limited to, forexample, any anti-PCSK9 (proprotein convertase subtilisin/kexin type 9)antibody, such as those described in US2010/0166768. Other anti-PCSK9antibodies are described in US2010/0040611, US2010/0041102,US2010/0040610, US2010/0113575, US2009/0232795, US2009/0246192,US2010/0233177, US2009/0142352, US2009/0326202, US2010/0068199,US2011/0033465, US2011/0027287, US2010/0150937, US2010/0136028 andWO2009/055783.

In certain embodiments, it may be beneficial to administer the anti-GCGRantibodies of the invention in combination with a nucleic acid thatinhibits the activity of PCSK9 (proprotein convertase subtilisin/kexintype 9), such as an antisense molecule, a double stranded RNA, or asiRNA molecule. Exemplary nucleic acid molecules that inhibit theactivity of PCSK9 are described in US2011/0065644, US2011/0039914,US2008/0015162 and US2007/0173473.

The additional therapeutically active component(s) may be administeredprior to, concurrent with, or after the administration of the anti-GCGRantibody of the present invention. For purposes of the presentdisclosure, such administration regimens are considered theadministration of an anti-GCGR antibody “in combination with” a secondtherapeutically active component.

Administration Regimens

According to certain embodiments of the present invention, multipledoses of one or more anti-GCGR antibodies (an antibody combination) or abi-specific antigen-binding molecule may be administered to a subjectover a defined time course. The methods according to this aspect of theinvention comprise sequentially administering to a subject multipledoses of an antibody, antibody combination, or a bi-specificantigen-binding molecule of the invention. As used herein, “sequentiallyadministering” means that each dose of an antibody, antibodycombination, or a bi-specific antigen-binding molecule is administeredto the subject at a different point in time, e.g., on different daysseparated by a predetermined interval (e.g., hours, days, weeks ormonths). The present invention includes methods, which comprisesequentially administering to the patient a single initial dose of anantibody, antibody combination, or a bi-specific antigen-bindingmolecule, followed by one or more secondary doses of the antibody, andoptionally followed by one or more tertiary doses of the antibody.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of an antibody, antibodycombination, or a bi-specific antigen-binding molecule of the invention.Thus, the “initial dose” is the dose which is administered at thebeginning of the treatment regimen (also referred to as the “baselinedose”); the “secondary doses” are the doses which are administered afterthe initial dose; and the “tertiary doses” are the doses which areadministered after the secondary doses. The initial, secondary, andtertiary doses may all contain the same amount of an antibody, antibodycombination, or a bi-specific antigen-binding molecule, but generallymay differ from one another in terms of frequency of administration. Incertain embodiments, however, the amount of an antibody, antibodycombination, or a bi-specific antigen-binding molecule contained in theinitial, secondary and/or tertiary doses varies from one another (e.g.,adjusted up or down as appropriate) during the course of treatment. Incertain embodiments, two or more (e.g., 2, 3, 4, or 5) doses areadministered at the beginning of the treatment regimen as “loadingdoses” followed by subsequent doses that are administered on a lessfrequent basis (e.g., “maintenance doses”).

In one exemplary embodiment of the present invention, each secondaryand/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2, 2½, 3, 3½,4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13,13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21,21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more) weeks afterthe immediately preceding dose. The phrase “the immediately precedingdose,” as used herein, means, in a sequence of multiple administrations,the dose of an antibody, antibody combination, or a bi-specificantigen-binding molecule, which is administered to a patient prior tothe administration of the very next dose in the sequence with nointervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an antibody, antibody combination, or a bi-specific antigen-bindingmolecule that specifically binds Fel d1. For example, in certainembodiments, only a single secondary dose is administered to thepatient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8,or more) secondary doses are administered to the patient. Likewise, incertain embodiments, only a single tertiary dose is administered to thepatient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8,or more) tertiary doses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks after the immediately preceding dose. Similarly, in embodimentsinvolving multiple tertiary doses, each tertiary dose may beadministered at the same frequency as the other tertiary doses. Forexample, each tertiary dose may be administered to the patient 2 to 4weeks after the immediately preceding dose. Alternatively, the frequencyat which the secondary and/or tertiary doses are administered to apatient can vary over the course of the treatment regimen. The frequencyof administration may also be adjusted during the course of treatment bya physician depending on the needs of the individual patient followingclinical examination.

Diagnostic Uses of the Antibodies

The anti-GCGR antibodies of the present invention may also be used todetect and/or measure GCGR in a sample, e.g., for diagnostic purposes.For example, an anti-GCGR antibody, or fragment thereof, may be used todiagnose a condition or disease characterized by aberrant expression(e.g., over-expression, under-expression, lack of expression, etc.) ofGCGR. Exemplary diagnostic assays for GCGR may comprise, e.g.,contacting a sample, obtained from a patient, with an anti-GCGR antibodyof the invention, wherein the anti-GCGR antibody is labeled with adetectable label or reporter molecule or used as a capture ligand toselectively isolate GCGR protein from patient samples. Alternatively, anunlabeled anti-GCGR antibody can be used in diagnostic applications incombination with a secondary antibody which is itself detectablylabeled. The detectable label or reporter molecule can be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent orchemiluminescent moiety such as fluorescein isothiocyanate, orrhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase,horseradish peroxidase, or luciferase. Specific exemplary assays thatcan be used to detect or measure GCGR in a sample include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), andfluorescence-activated cell sorting (FACS).

Samples that can be used in GCGR diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient, which contains detectable quantities of GCGR protein, orfragments thereof, under normal or pathological conditions. Generally,levels of GCGR in a particular sample obtained from a healthy patient(e.g., a patient not afflicted with a disease or condition associatedwith abnormal GCGR levels or activity) will be measured to initiallyestablish a baseline, or standard, level of GCGR. This baseline level ofGCGR can then be compared against the levels of GCGR measured in samplesobtained from individuals suspected of having a GCGR related disease orcondition, or symptoms associated with such disease or condition.

EXAMPLES

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, preferred methods and materials are now described. Allpublications mentioned herein are incorporated herein by reference intheir entirety.

Example 1 Generation of Human Antibodies to Human GCGR

An immunogen comprising any one of the following can be used to generateantibodies to hGCGR. For example, cells expressing hGCGR were used incertain embodiments as an immunogen to generate antibodies to hGCGR.Additionally, DNA encoding hGCGR was used in certain embodiments as animmunogen to prepare the antibodies of the invention. Furthermore, incertain embodiments, peptides comprising amino acid sequences from theN-terminal domain of hGCGR were utilized as an immunogen to generateantibodies to human GCGR. In addition, in certain embodiments, peptidescomprising amino acid sequences from any of the extracellular loopregions EC1, EC2, or EC3, of hGCGR may be utilized as an immunogen togenerate antibodies to human GCGR. The cells, DNA, or peptides that wereused as immunogens, as noted above, were administered directly, with anadjuvant to stimulate the immune response, to a VELOCIMMUNE® mousecomprising DNA encoding human Immunoglobulin heavy and kappa light chainvariable regions. The antibody immune response was monitored by aGCGR-specific immunoassay. When a desired immune response was achievedsplenocytes were harvested and fused with mouse myeloma cells topreserve their viability and form hybridoma cell lines. The hybridomacell lines were screened and selected to identify cell lines thatproduce GCGR-specific antibodies. Using this technique, and the variousimmunogens described above, several anti-GCGR chimeric antibodies (i.e.,antibodies possessing human variable domains and mouse constant domains)were obtained; certain exemplary antibodies generated in this mannerwere designated as H4H1345N, H4H1617N and H4H1765N.

Anti-GCGR antibodies were also isolated directly from antigen-positive Bcells without fusion to myeloma cells, as described in U.S.2007/0280945A1, herein specifically incorporated by reference in itsentirety. Using this method, several fully human anti-GCGR antibodies(i.e., antibodies possessing human variable domains and human constantdomains) were obtained; exemplary antibodies generated in this mannerwere designated as follows: H4H1321B, H4H1321P, H4H1327B, H4H1327P,H4H1328B, H4H1328P, H4H1331B, H4H1331P, H4H1339B and H4H1339P.

The biological properties of the exemplary anti-GCGR antibodiesgenerated in accordance with the methods of this Example are describedin detail in the Examples set forth below.

Example 2 Heavy and Light Chain Variable Region Amino Acid Sequences

Table 1 sets forth the heavy and light chain variable region amino acidsequence pairs of selected anti-GCGR antibodies and their correspondingantibody identifiers. Antibodies having the same numerical antibodydesignation, but differing by a letter suffix of N, B or P refer toantibodies having heavy and light chains with identical CDR sequencesbut with sequence variations in regions that fall outside of the CDRsequences (i.e., in the framework regions). Thus, N, B and P variants ofa particular antibody have identical CDR sequences within their heavyand light chain variable regions but differ from one another withintheir framework regions.

TABLE 1 Antibody SEQ ID NOs: Designation HCVR HCDR1 HCDR2 HCDR3 LCVRLCDR1 LCDR2 LCDR3 H4H1345N 2 4 6 8 10 12 14 16 H4H1617N 18 20 22 24 2628 30 32 H4H1765N 34 36 38 40 42 44 46 48 H4H1321B 50 52 54 56 58 60 6264 H4H1321P 66 52 54 56 68 60 62 64 H4H1327B 70 72 74 76 78 80 82 84H4H1327P 86 72 74 76 88 80 82 84 H4H1328B 90 92 94 96 98 100 102 104H4H1328P 106 92 94 96 108 100 102 104 H4H1331B 110 112 114 116 118 120122 124 H4H1331P 126 112 114 116 128 120 122 124 H4H1339B 130 132 134136 138 140 142 144 H4H1339P 146 132 134 136 148 140 142 144

Example 3 Variable Gene Utilization Analysis

To analyze the structure of antibodies produced, the nucleic acidsencoding antibody variable regions were cloned and sequenced. From thenucleic acid sequence and predicted amino acid sequence of theantibodies, gene usage was identified for each Heavy Chain VariableRegion (HCVR) and Light Chain Variable Region (LCVR). Table 2 sets forththe gene usage for selected antibodies in accordance with the invention.

TABLE 2 Antibody Identifier HCVR/LCVR HCVR LCVR Antibody SEQ ID NOsV_(H) D_(H) J_(H) V_(K) J_(K) H4H1617N 18/26 V1-24 D3-9 J6 V2-28 J1H4H1345N  2/10 V1-24 D3-9 J6 V2-28 J1 H4H1765N 34/42 V3-48 D6-6 J6 V2-28J1 H4H1321P 66/68 V3-30 D3-9 J6 V1-16 J4 H4H1327P 86/88 V3-7  D3-9 J6V1-17 J3 H4H1328P 106/108 V3-13 D3-9 J6 V1-17 J4 H4H1331P 126/128 V3-33D3-9 J6 V1-17 J1/J4 H4H1339P 146/148 V3-13 D3-9 J6 V1-6  J1

Example 4 Antibody Binding to Soluble GCGR as Determined by SurfacePlasmon Resonance

Binding affinities and kinetic constants of human monoclonal anti-hGCGRantibodies binding to human and monkey soluble recombinant hGCGRectodomain (hGCGR and MfGCGR, respectively) were determined by surfaceplasmon resonance at both 25° C. and 37° C. Measurements were conductedon a T100 Biacore instrument. Antibodies were captured onto the Biacoresensor chip surface via a covalently-linked anti-human kappa antibodycapture surface, and the soluble GCGR proteins were applied to thesurface either in a monovalent (hGCGR expressed with amyc-myc-hexa-histidine C-terminal tag) or bivalent (hGCGR and MfGCGRexpressed with an N-terminal Fc fusion) format. The amino acid sequenceidentifiers of the reagents used in this example are shown in Table 3.

TABLE 3 Description Construct SEQ ID NO: anti-GCGR positive 150 kDa,dimer (See Yan, Hai et al. control hIgG4(S108P) WO2008/036341)mfGCGR-N-terminal 160k 152 hFc hGCGR-mFc 80654.42 Da, dimer 149hGCGR-mmh 18,965 Da, monomer 151

The soluble GCGR was applied to the flow cell in separate injections atmultiple concentrations ranging from 3.1 nM to 50 nM, and kineticassociation (k_(a)) and dissociation (k_(d)) rate constants weredetermined by fitting the data to a 1:1 binding model using Scrubberv2.0a curve fitting software. Binding dissociation equilibrium constantsand dissociative half-lives were calculated from the kinetic rateconstants as: K_(D)=k_(d)/k_(a); t_(1/2)=(In2/k_(d)).

TABLE 4a Biacore data for binding at 25° C. Antibody Antigen T½Designation tested ka kd K_(D) (min) H4H1321P hGCGR-mmh 1.06E+063.74E−03 3.54E−09 3 hGCGR-hFc 1.20E+06 2.85E−04 2.38E−10 41 mfGCGR-hFc1.76E+06 4.18E−05 2.38E−11 276 H4H1327P hGCGR-mmh 9.52E+05 3.52E−043.69E−10 33 hGCGR-hFc 1.21E+06 4.10E−05 3.38E−11 282 mfGCGR-hFc 1.60E+061.44E−05 9.02E−12 802 H4H1328P hGCGR-mmh 1.03E+06 2.12E−03 2.06E−09 5hGCGR-hFc 1.13E+06 2.26E−04 2.01E−10 51 mfGCGR-hFc 1.60E+06 8.46E−055.29E−11 137 H4H1331P hGCGR-mmh 6.57E+05 1.11E−04 1.70E−10 104 hGCGR-hFc7.60E+05 1.54E−05 2.02E−11 751 mfGCGR-hFc 1.17E+06 8.12E−06 6.90E−121423 H4H1339P hGCGR-mmh 6.45E+05 5.32E−04 8.25E−10 22 hGCGR-hFc 1.00E+066.20E−05 6.18E−11 186 mfGCGR-hFc 1.26E+06 2.28E−05 1.82E−11 506 H4H1345NhGCGR-mmh 7.98E+05 3.44E−04 4.31E−10 34 hGCGR-hFc 7.90E+05 5.72E−057.24E−11 202 mfGCGR-hFc 9.53E+05 2.42E−05 2.54E−11 477 H4H1617NhGCGR-mmh 1.07E+06 1.99E−04 1.87E−10 58 hGCGR-hFc 8.18E+05 3.18E−053.89E−11 363 mfGCGR-hFc 1.26E+06 1.38E−05 1.10E−11 835 H4H1765NhGCGR-mmh 3.26E+05 3.05E−05 9.30E−11 379 hGCGR-hFc 4.10E+05 4.95E−061.22E−11 2331 mfGCGR-hFc 6.43E+05 1.00E−06 1.56E−12 11550 Isotype-hGCGR-mmh 6.67E+05 1.68E−04 2.52E−10 69 matched hGCGR-hFc 8.21E+052.04E−05 2.49E−11 565 comparator mfGCGR-hFc 1.23E+06 6.09E−06 4.95E−121897 antibody

TABLE 4b Biacore data for binding at 37° C. Antibody Antigen T½Designation tested ka kd K_(D) (min) H4H1321P hGCGR-mmh 1.58E+062.02E−02 1.28E−08 1 hGCGR-hFc 1.41E+06 1.09E−04 7.70E−11 106 mfGCGR-hFc2.19E+06 7.59E−05 3.47E−11 152 H4H1327P hGCGR-mmh 1.48E+06 2.00E−031.35E−09 6 hGCGR-hFc 1.48E+06 2.32E−04 1.57E−10 50 mfGCGR-hFc 2.22E+067.94E−05 3.57E−11 145 H4H1328P hGCGR-mmh 1.61E+06 1.08E−02 6.67E−09 1hGCGR-hFc 1.55E+06 1.92E−04 1.24E−10 60 mfGCGR-hFc 2.03E+06 7.17E−053.53E−11 161 H4H1331P hGCGR-mmh 9.73E+05 5.19E−04 5.33E−10 22 hGCGR-hFc1.17E+06 9.12E−05 7.79E−11 127 mfGCGR-hFc 1.60E+06 4.12E−05 2.57E−11 281H4H1339P hGCGR-mmh 8.76E+05 4.30E−03 4.91E−09 3 hGCGR-hFc 1.17E+063.71E−04 3.18E−10 31 mfGCGR-hFc 1.69E+06 1.07E−04 6.31E−11 108 H4H1345NhGCGR-mmh 9.28E+05 1.97E−03 2.12E−09 6 hGCGR-hFc 9.52E+05 3.09E−043.24E−10 37 mfGCGR-hFc 1.27E+06 1.28E−04 1.01E−10 91 H4H1617N hGCGR-mmh1.20E+06 1.13E−03 9.43E−10 10 hGCGR-hFc 1.18E+06 2.14E−04 1.81E−10 54mfGCGR-hFc 1.49E+06 8.72E−05 5.86E−11 133 H4H1765N hGCGR-mmh 4.41E+051.11E−04 2.52E−10 104 hGCGR-hFc 6.64E+05 3.57E−05 5.37E−11 324mfGCGR-hFc 9.04E+05 1.48E−05 1.64E−11 778 Isotype- hGCGR-mmh 8.73E+051.46E−03 1.68E−09 8 matched hGCGR-hFc 1.15E+06 1.82E−04 1.59E−10 63comparator mfGCGR-hFc 1.66E+06 6.27E−05 3.77E−11 184 antibody

As shown in Tables 4a and 4b, the exemplary antibodies exhibited highaffinity binding to both human and monkey GCGR soluble proteins. Asignificant increase in binding affinity (5-fold to 15-fold) wasobserved when flowing the bivalent hGCGR in comparison to monovalenthGCGR. The antibodies consistently bound with higher affinity (3-fold to10-fold) to the monkey variant, MfGCGR, compared to hGCGR.

Example 5 Bioassay to Measure the Effects of Anti-GCGR Antibodies onGCGR Activation

GCGR is a G-protein coupled receptor and its ligand, glucagon (GCG),stimulates adenylyl cyclase activity through Gαs and phosphoinositolturnover through Gq (Jiang and Zhang, (2003), Am J Physiol EndocrinolMetab 284: E671-E678). A bioassay was developed to detect activationthrough Gαs, subsequent elevation of cAMP levels and transcriptionalactivation. HEK293 cell lines were generated to stably expressfull-lengths of human GCGR (GenBank accession number NP_(—)000151.1; SEQID NO: 153), monkey (Macaca fascicularis) GCGR (SEQ ID NO: 155), andmouse GCGR (NP_(—)032127.2; SEQ ID NO: 154) along with a luciferasereporter assay. The stable cell lines were isolated and maintained in10% FBS, DMEM, NEAA, Pen/Strep, and 500 mg/ml G418. For rat GCGR, theHEK293 cell line expressing the reporter gene [CRE(4×)-luciferase-IRES-GFP] was transiently transfected with full-lengthrat GCGR (NP_(—)742089.1; SEQ ID NO: 156) using Lipofectamine-2000(Invitrogen).

For the bioassay, 293/GCGR cells were seeded onto 96-well assay platesat 20,000 cells/well in low serum media, 0.1% FBS and OPTIMEM®, andincubated at 37° C. and 5% CO₂ overnight. Next day, GCG was seriallydiluted at 1:3 and added to cells starting from 100 nM to 0.002 nMincluding no GCG control for dose response. For inhibition, antibodieswere serially diluted at 1:3 and added to cells starting from 200 to0.003 (for hGCGR cells) or 100 nM to 0.002 nM (for monkey, mouse and ratGCGR cells) including no antibody control with constant concentration of100 pM GCG. Luciferase activity was detected after 5.5 hrs of incubationin 37° C. and 5% CO₂.

EC50 values for stimulation of each reporter cell-line by 100 pM GCG areshown in Table 5a. The results of the IC50 values for antibodiesblocking stimulation of cells by 100 pM GCG are shown in Table 5b,including the results for two control antibodies, Control mAb1 (positivecontrol expressed as hlgG4 isotype; for example, see WO2008/036341 forthe antibody designated as “A-9” having the HCVR of SEQ ID NO: 275,HCDR1 of SEQ ID NO: 102, HCDR2 of SEQ ID NO: 128, and HCDR3 of SEQ IDNO: 169 and the LCVR of SEQ ID NO: 229, LCDR1 of SEQ ID NO: 14, LCDR2 ofSEQ ID NO: 50 and the LCDR3 of SEQ ID NO: 74) and Control mAb2 (anisotype-matched negative control).

Regarding the inhibition of human GCGR by anti-GCGR antibodies, theactivation of GCGR by GCG was shown to stimulate luciferase activitywith an EC50 of 113 pM and all antibodies except Control mAb2 (isotypematched negative control) blocked the activation of GCG at 100 pM anddecreased the luciferase activity.

With respect to the inhibition of monkey GCGR by anti-GCGR antibodies,the activation of GCGR by GCG was shown to stimulate luciferase activitywith an EC50 of 36 pM and all antibodies except Control mAb2 (isotypematched negative control) blocked the activation of GCG at 100 pM anddecreased the luciferase activity. H4H1765N showed a partial inhibitionof GCG at highest concentration of antibody tested, 100 nM.

Regarding the inhibition of mouse GCGR by anti-GCGR antibodies, theactivation of GCGR by GCG was shown to stimulate luciferase activitywith an EC50 of 83 pM and all antibodies except H4H1345N, H4H1617N,H4H1765N and Control mAb2 (isotype matched negative control) blocked theactivation of GCG at 100 pM and decreased the luciferase activity.

With respect to the inhibition of rat GCGR by anti-GCGR antibodies, theactivation of GCGR by GCG was shown to stimulate luciferase activitywith an EC50 of 252 pM and all antibodies except H4H1765N and ControlmAb2 (isotype matched negative control) blocked the activation of GCG at100 pM and decreased the luciferase activity.

TABLE 5a Cell lines hGCGR mfGCGR mGCGR rat GCGR EC50 (pM) 113 36 83 252Constant GCG (pM) 100

TABLE 5b Antibody IC50 (nM) Designation hGCGR mfGCGR mGCGR rat GCGRH4H1321P 0.27 4.03 1.13 1.21 H4H1327P 0.39 2.56 1.04 0.88 H4H1328P 0.242.73 1.26 0.93 H4H1331P 0.66 8.29 1.62 3.87 H4H1339P 0.46 2.85 1.60 0.97H4H1345N 2.22 3.86 Not 8.07 Blocked H4H1617N 1.25 4.24 Not 3.66 BlockedH4H1765N 12.78 75.16 Not Not Blocked Blocked Control mAb1 0.30 2.38 1.690.69 Positive control Control mAb2 Not Not Not Not Negative controlBlocked Blocked Blocked Blocked

In summary, eight anti-hGCGR fully-human antibodies were tested anddemonstrated blocking of activation of human GCGR by 100 pM GCG in areporter cell line that exhibited an EC50 of 113 pM when stimulated byGCG alone. In the monkey GCGR reporter cell line, seven out of eighttested antibodies fully inhibited activation by 100 pM GCG. H4H1765N didnot fully inhibit monkey GCGR at the highest concentration of antibodytested, 100 nM. Five out of eight of the antibodies fully inhibited theactivation by 100 pM GCG in the mouse GCGR reporter cell line, and sevenout of eight antibodies inhibited the activation by 100 pM GCG in therat GCGR-transfected reporter cells.

Example 6 Effect of Anti-GCGR Antibodies in ob/ob Mice

Selected anti-hGCGR antibodies, all of which cross-react with mouseGCGR, were tested for their ability to reduce blood glucose levels inob/ob mice, a mouse model of type 2 diabetes. ob/ob mice were put intoten groups of five or six animals. Each group received subcutaneousinjections of each antibody at 1 or 10 mg/kg. The control group wasinjected with a hlgG isotype control antibody, which does not bind toany known mouse proteins. Two or seven days after antibody dosing at 1or 10 mg/kg, respectively, a few drops of blood obtained by tail bleedswere collected from mice. Specifically, for the group given the antibodydesignated H4H1327P at 10 mg/kg, tail bleeds were collected morefrequently at 2, 4, 7, 9, 11, 14, 16, 18, and 21 days after dosing.Blood glucose levels from the tail bleed samples were determined byACCU-CHEK® Compact Plus (Roche). The percent reduction in blood glucosefrom the mean blood glucose levels of the control group was calculatedfor each animal at each time point. The average percent reduction inblood glucose was calculated for each antibody group. Table 6 summarizesthe mean blood glucose levels of the control group. Results, expressedas (mean±SEM) of percent blood glucose reduction, are shown in Tables 7aand 7b.

TABLE 6 Time Blood glucose (mg/dL) Day 0 197 ± 14 Day 2 185 ± 13 Day 4167 ± 6  Day 7 202 ± 20 Day 9 205 ± 18 Day 11 195 ± 23 Day 14 229 ± 13Day 16 206 ± 6  Day 18 187 ± 11 Day 21 209 ± 16

TABLE 7a Blood glucose reduction (%) Time Antibody Designation Dosage(days) H4H1327P H4H1328P H4H1331P H4H1339P  1 mg/kg 2 49 ± 1 45 ± 2 46 ±2 46 ± 2 10 mg/kg 7 53 ± 2 50 ± 2 55 ± 2 52 ± 2

TABLE 7b Blood glucose reduction (%) Antibody Time (days) Designation 24 7 9 11 14 16 18 21 H4H1327P 58 ± 2 52 ± 2 53 ± 2 56 ± 2 47 ± 3 51 ± 345 ± 4 34 ± 5 −5 ± 17

Mice treated with the anti-hGCGR antibodies tested (shown in Tables 7aand 7b) exhibited significant reductions in blood glucose levelscompared to mice injected with control antibody.

Example 7 Effect of Anti-GCGR Antibodies in Transgenic Mice Expressingthe Human GCGR Protein

The effects of anti-hGCGR antibodies on blood glucose and plasma lipidlevels were determined in transgenic mice expressing the human GCGRprotein (“humanized GCGR mice”). Humanized GCGR mice were generated byreplacing the mouse GCGR gene with the human GCGR gene (SEQ ID NO: 157;encoding full-length protein, GenBank accession number NP_(—)000151.1;SEQ ID NO: 153) in C57BL6/129 (F1H4) embryonic stem cells. After germline transmission was established, heterozygous mice (GCGR^(hum/+)) werebred together to generate homozygous mice (GCGR^(hum/hum)) on a C57BL6background. Homozygous humanized GCGR mice were put into ten groups ofthree or four animals. Each group received subcutaneous injections ofeach antibody at 3 mg/kg. The Control I group was injected with a hlgGisotype negative control antibody, which does not bind to any knownmouse proteins. The Control II group was injected with an anti-hGCGRhlgG4 positive control/comparator antibody (See WO2008/036341 for the“A9” antibody sequence), which has been validated to decrease bloodglucose levels of humanized GCGR mice. Mice were bled three days afterantibody dosing, and blood glucose levels were determined by ACCU-CHEK®Compact Plus (Roche). The percent reduction in blood glucose from themean blood glucose levels of the Control I group was calculated for eachanimal. The average percent reduction in blood glucose was calculatedfor each antibody group. Table 8a summarizes the mean blood glucoselevels of the negative control group. Results, expressed as (mean±SEM)of percent blood glucose reduction in animals treated with either thepositive Control II comparator antibody, or the test anti-GCGRantibodies as compared to the negative control group, are shown in Table8b.

Additionally with the Control I, Control II and H4H1765N groups, micewere bled before and 3 and 8 days after antibody dosing, and plasmalipid levels were determined by ADVIA® 1650 Chemistry System (Siemens).Averages were calculated for each of the measurements of low densitylipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol(HDL-C), total cholesterol (TOTAL-C), triglycerides (TG), nonesterifiedfatty acids (NEFA) levels for each of the three groups. Results,expressed as (mean±SEM) of plasma lipid concentrations, are shown inTable 8c.

Mice treated with most of the anti-hGCGR antibodies tested (shown inTable 8b) exhibited significant reductions in blood glucose levelssimilar to mice receiving the positive control II antibody. Mice treatedwith certain of the anti-hGCGR antibodies tested (e.g. H4H1765N, shownin Table 8c) exhibited significant reductions in triglyceride levelscompared to mice receiving negative Control I antibody. In particular,the lowering of triglyceride levels was observed with two anti-GCGRantibodies, one designated as H4H1765N (data shown below in Table 8c)and the other designated as H4H1327P (data not shown).

TABLE 8a Time Blood glucose (mg/dL) Day 0 155 ± 6 Day 1 160 ± 5 Day 3155 ± 5 Day 6 164 ± 7 Day 8 156 ± 7 Day 10 154 ± 9 Day 13 149 ± 5

TABLE 8b Antibody Designation Blood glucose reduction (%) Control II 37± 3 H4H1321P 32 ± 4 H4H1327P 40 ± 7 H4H1328P 31 ± 7 H4H1331P 33 ± 4H4H1339P 31 ± 6 H4H1617N 15 ± 5 H4H1345N 14 ± 2 H4H1765N 32 ± 4

TABLE 8c Antibody Time LDL-C HDL-C TOTAL-C TG NEFA Designation (days)(mg/dL) (mg/dL) (mg/dL) (mg/dL) (mmol/L) Control I Pre 9.4 ± 1.4 47 ± 4 97 ± 10 98 ± 6 0.67 ± 0.06 3 7.7 ± 1.1 45 ± 3 95 ± 4 80 ± 8 0.96 ± 0.038 9.8 ± 2.2 46 ± 2 99 ± 4 120 ± 19 0.88 ± 0.05 Control II Pre 6.7 ± 0.437 ± 1 76 ± 2  69 ± 12 0.60 ± 0.09 3 11.2 ± 1.6  51 ± 2 101 ± 8  58 ± 50.80 ± 0.11 8 14.4 ± 2.1  57 ± 3 114 ± 7   74 ± 15 0.73 ± 0.04 H4H1765NPre 8.7 ± 0.2 39 ± 6 79 ± 8  94 ± 19 0.81 ± 0.12 3 8.0 ± 1.1 46 ± 6 91 ±9 68 ± 7 0.72 ± 0.06 8 9.2 ± 1.6 46 ± 6 92 ± 8 75 ± 7 0.70 ± 0.05

Example 8 Effect of Combination Therapy with an Anti-GCGR Antibody andan Antibody Specific for PCSK9 (Proprotein Convertase Subtilisin/KexinType 9) on Blood Glucose, Plasma Lipid and Hepatic Triglyceride Levelsin Mice Reagents

The following antibodies were used to study the effect of combinationtherapy with an anti-GCGR antibody and an antibody specific for PCSK9 onblood glucose levels, plasma lipid levels and hepatic triglyceridelevels in C57BL/6 mice: An anti-hIL4R antibody designated REGN496, whichis an hlgG4 isotype control; an anti-GCGR (hlgG4) antibody designatedH4H1327P; and an anti-PCSK9 (hlgG1) antibody designated H1H316P. Theamino acid sequence identifiers for the HCVR, LCVR, HCDRs, and LCDRs areshown below in Table 9.

TABLE 9 REGN AB SEQ ID NUMBERS Designation HCVR HCDR1 HCDR2 HCDR3 LCVRLCDR1 LCDR2 LCDR3 REGN496 194 195 196 197 198 199 200 201 H4H1327P 86 7274 76 88 80 82 84 H1H316P 173 161 163 165 175 167 169 171

EXPERIMENTAL PROCEDURE

The combined effects of H4H1327P, an anti-hGCGR antibody, and H1H316P,an anti-hPCSK9 antibody, on blood glucose, plasma lipid, and hepatictriglyceride (TG) levels were determined in C57BL/6 mice.

H4H1327P cross-reacts with mouse GCGR, and H1H316P cross-reacts withmouse PCSK9. C57BL/6 mice were put into six groups of six animals. Eachgroup received once a week subcutaneous injections of an antibody or acombination of two antibodies. The first group was injected at 10 mg/kgwith a hlgG4 isotype control antibody, which does not bind to any knownmouse protein. The second and third group received H4H1327P at 3 mg/kgand 10 mg/kg, respectively. The fourth group was injected with 10 mg/kgH1H316P. The fifth group received a combination of 3 mg/kg H4H1327P and10 mg/kg H1H316P, and the sixth group was injected with a combination of10 mg/kg H4H1327P and 10 mg/kg H1H316P. Eleven and 19 days after theinitial antibody dosing, mice were bled for blood glucose and plasmalipid measurements. At Day 19, liver was harvested for the determinationof hepatic TG content. Blood glucose levels were measured with the useof ACCU-CHEK® Compact Plus (Roche). The percent reduction in bloodglucose from the mean blood glucose level of the isotype control groupwas calculated for each animal. The percent reduction and associatederror in blood glucose for each treatment group was then calculated byaveraging across values for the individual animals in each group.Results, expressed as (mean±SEM) of percent blood glucose reduction, areshown in Table 10a and in FIG. 1.

Plasma lipid levels were determined by ADVIA® 1650 Chemistry System(Siemens). Hepatic TG contents were measured using a colorimetric assay(Teco Diagnostics) after extraction of TG from tissue withchloroform/methanol. Means were calculated for each of the measurementsof plasma low density lipoprotein cholesterol (LDL-C), high densitylipoprotein cholesterol (HDL-C), total cholesterol (TOTAL-C), TG andhepatic TG levels for each group. Results, expressed as (mean±SEM) ofplasma and hepatic lipid concentrations, are shown in Table 10b and inFIG. 2 (plasma LDL-C levels); FIG. 3 (Plasma HDL-C levels); FIG. 4(Plasma Total-C levels); FIG. 5 (Plasma TG levels); and FIG. 6 (hepaticTG content).

Results Summary and Conclusions:

TABLE 10a Blood glucose reduction (%) Time H4H1327P H4H1327P H1H316PH4H1327P 3 mg/kg + H4H1327P 10 mg/kg + (days) 3 mg/kg 10 mg/kg 10 mg/kgH1H316P 10 mg/kg H1H316P 10 mg/kg 11 33 ± 7 40 ± 5 3 ± 3 28 ± 7 39 ± 219 25 ± 4 28 ± 4 −4 ± 6  21 ± 7 27 ± 2

TABLE 10b Plasma Liver TG LDL-C HDL-C TOTAL-C TG (mg/g Antibody Time(days) (mg/dL) (mg/dL) (mg/dL) (mg/dL) tissue) Control Pre-dosing 5.2 ±0.7 37 ± 2 65 ± 2 101 ± 11 NA 11 4.4 ± 0.3 39 ± 2 66 ± 2  72 ± 16 NA 196.0 ± 0.3 46 ± 1 74 ± 1 84 ± 6 2.4 ± 0.1 H4H1327P Pre-dosing 3.3 ± 0.135 ± 1 58 ± 2 109 ± 11 NA 3 mg/kg 11 7.4 ± 0.6 51 ± 2 89 ± 3  96 ± 14 NA19 7.7 ± 0.2 59 ± 4 93 ± 5 80 ± 8 2.4 ± 0.2 H4H1327P Pre-dosing 4.2 ±0.2 39 ± 1 67 ± 2 120 ± 16 NA 10 mg/kg 11 8.3 ± 0.3 54 ± 3 91 ± 4 70 ± 8NA 19 10.4 ± 0.8  56 ± 1 95 ± 3 75 ± 5 1.9 ± 0.1 H1H316P Pre-dosing 4.0± 0.2 35 ± 2 62 ± 3 114 ± 8  NA 10 mg/kg 11 3.0 ± 0.3 21 ± 3 48 ± 5 97 ±8 NA 19 2.9 ± 0.2 30 ± 1 50 ± 1  91 ± 11 2.1 ± 0.3 H4H1327P Pre-dosing4.2 ± 0.2 36 ± 1 64 ± 1 124 ± 12 NA 10 mg/kg + 11 4.3 ± 0.3 38 ± 1 65 ±1 109 ± 16 NA H1H316P 19 5.4 ± 0.5 40 ± 2 66 ± 3  96 ± 11 2.1 ± 0.1 10mg/kg H4H1327P Pre-dosing 4.8 ± 0.7 38 ± 2 65 ± 4 107 ± 18 NA 3 mg/kg +11 4.9 ± 0.5 41 ± 1 67 ± 2  89 ± 10 NA H1H316P 19 5.3 ± 0.6 42 ± 2 67 ±3 85 ± 9 2.1 ± 0.2 10 mg/kg NA: Not applicable

Tabulated Data Summary:

Mice treated with H4H1327P alone showed significant reductions in bloodglucose levels and increases in LDL, HDL, and total cholesterol levelsin comparison to mice receiving control mAb. Mice treated with H1H316Palone exhibited significant reductions in cholesterol levels with nochange in blood glucose levels. Mice treated with a combination ofH4H1327P and H1H316P showed significant reductions in blood glucoselevels with no alterations in cholesterol levels in comparison to micereceiving control mAb. Hepatic TG contents were not altered in any ofthe treatment groups compared to the isotype control group.

Example 9 The Effect of an Anti-GCGR Antibody in a Diet-Induced ObesityMouse Model

The effects of H4H1327P, an anti-hGCGR antibody that cross-reacts withmouse GCGR, on blood glucose levels and body weights were determined ina diet-induced obesity (D10) mouse model of type 2 diabetes (T2D).

The DIO model is developed by feeding mice on a high fat (60% kcal) diet(HFD) from 5-6 weeks of age. After approximately 6 weeks on the diet,mice develop metabolic abnormalities including insulin resistance,glucose intolerance, and obesity. The DIO model induces a physiologicalcondition in mice closer to human T2D than that induced by the other twocommonly used T2D models, ob/ob and db/db mice, since the latter twomodels result from mutations in leptin or leptin receptor genes,respectively, which are rarely the cause of T2D in humans.

In this study, eleven week-old male C57BL/6 mice, placed on HFD since 5weeks of age, were purchased from Taconic farms, Inc. and kept on thediet for another 8 weeks. The mice were divided into 4 groups of 10animals per group at 10 weeks of age. Each group received weekly (ondays 0, 7, 14, and 21) subcutaneous injections of H4H1327P at 3, 10, or30 mg/kg, or 30 mg/kg of the hlgG4 isotype control, which does not bindto any known mouse protein. Blood glucose levels and body weights weremeasured periodically, and 6 days after administering the last antibodydose (on day 27), 6 mice/group were sacrificed. For the next 6 weeks,blood glucose and body weights were monitored for the remaining 4mice/group. The percent reduction in blood glucose levels compared tothe mean blood glucose level of the isotype control group was calculatedfor each animal. The percent reduction and associated error in bloodglucose for each treatment group was then calculated by averaging acrossvalues for the individual animals in each group. Results, expressed as(mean±SEM) of percent blood glucose reduction, are shown in Table 11.The percent change in body weight from the baseline (weight at day 0)was calculated for each animal. The percent change and associated errorin body weight for each treatment group was then calculated by averagingacross values for the individual animals in each group. Results,expressed as (mean±SEM) of percent body weight change from baseline, areshown in Table 12.

Results Summary and Conclusions:

H4H1327P, at all dosages tested, reduced blood glucose and body weightof DIO mice compared to the isotype control groups. The greatestrelative blood glucose reduction (48.5%) occurred in the highest dose(30 mg/kg) group at day 10, and the greatest relative body weightreduction (12.8%) occurred in the highest dose group at day 28. Thelowest dose (3 mg/kg) groups achieved mean relative blood glucoselowering and mean body weight lowering values of at least 70% the valuesexhibited by the highest dose groups through day 28 (one week followingthe last dose). The observed blood glucose and body weight loweringeffects following the last treatment on day 21 (i.e., on days 28, 47,and 68) persisted longer for the higher H4H1327P dose groups compared tothe lower dose groups.

TABLE 11 Blood glucose reduction (%) Time H4H1327P H4H1327P H4H1327P(days) 3 mg/kg 10 mg/kg 30 mg/kg 10 44.6 ± 2.8 44.4 ± 1.8 48.5 ± 1.5 1933.9 ± 2.3 39.0 ± 1.9 37.5 ± 2.5 28 24.8 ± 1.7 28.1 ± 2.8 32.4 ± 1.6 47−3.9 ± 7.1 21.2 ± 7.0 30.0 ± 2.4 68 −4.3 ± 7.9 −1.1 ± 5.8 −12.9 ± 12.1

TABLE 12 Body weight change from baseline (%) Isotype Time controlH4H1327P H4H1327P H4H1327P (days) 30 mg/kg 3 mg/kg 10 mg/kg 30 mg/kg 101.8 ± 0.6 −5.5 ± 0.6 −6.1 ± 0.6 −5.8 ± 0.6 19 2.2 ± 0.6 −7.3 ± 0.6 −9.6± 0.6 −10.3 ± 0.7  28 1.8 ± 0.4 −9.2 ± 1.2 −10.5 ± 0.5  −12.8 ± 1.0  476.6 ± 0.8  5.2 ± 1.3  2.5 ± 1.4 −3.7 ± 2.2 68 10.4 ± 1.1   9.7 ± 0.710.6 ± 1.0  8.2 ± 1.1

Example 10 The Effect of an Anti-GCGR Antibody in a Streptozotocin(STZ)-Induced Mouse Model of Diabetic Ketoacidosis

The effects of H4H1327P, an anti-hGCGR antibody, which cross-reacts withmouse GCGR, on plasma ketone and glucose levels were determined in astreptozotocin (STZ)-induced mouse model of diabetic ketoacidosis (DKA).STZ is a chemical toxic to pancreatic beta cells of mammals which,therefore, destroys this cell type when administered to rodents. Asingle, high dose (200 mg/kg) injection of STZ to C57BL/6 mice leads tothe development of severe hyperglycemia and ketonuria, conditionsexhibited in human DKA, in 3 days. Nine-week-old male C57BL/6 mice,purchased from Taconic farms, Inc. were divided into 2 groups of 10animals, and each group received either intraperitoneal injections ofSTZ (in citrate buffer) at 200 mg/kg or vehicle (also in citratebuffer). Three days later, severe hyperglycemia (blood glucoselevels >400 mg/dL) and ketonuria were confirmed in all STZ treatedanimals. The next morning, the STZ treated mice were divided into 2groups of 5 animals, and each group received an intravenous injection ofH4H1327P or hlgG4 isotype control at 10 mg/kg. The citrate buffertreated mice were also divided into 2 groups of 5 animals, and eachgroup received intravenous injection of H4H1327P or hlgG4 isotypecontrol at 10 mg/kg. Mice were bled 18 hours before antibody dosing (2.5days after the STZ administration) and 28 hours after antibody dosingunder isoflurane anesthesia for plasma collection. Plasma ketone levelswere determined by beta-hydroxybutyrate assay (Biovision), and plasmaglucose levels by ADVIA® 1650 Chemistry System (Siemens). Averages werecalculated for the measurements of beta-hydroxybutyrate and glucoselevels for each of the four groups. Results, expressed as (mean±SEM) ofplasma beta-hydroxybutyrate and glucose concentrations, are shown inTable 13.

Results Summary and Conclusions:

A reduction (average 67%) in plasma beta-hydroxybutyrate (ketone)concentration was observed in STZ-induced diabetic ketoacidotic mice 28hours after H4H1327P treatment in comparison to plasma levels 18 hoursprior to the treatment, demonstrating that H4H1327P effectively loweredplasma ketone levels in a mouse model of DKA. For the STZ-treated micedosed with isotype control antibody, a 14% average increase in bloodglucose was observed for the serum samples collected 28 hours aftercontrol antibody treatment compared to the samples collected 18 hoursbefore antibody treatment, whereas for the H4H1327P dosed mice in theSTZ-treated group less than 1% average change in glucose was observedbetween serum samples collected at these two time points.

TABLE 13 Time from antibody Vehicle/ STZ/ treatment Isotype Vehicle/Isotype STZ/ (hrs) control H4H1327P control H4H1327P Beta- −18 1.1 ± 0.60.6 ± 0.2 3.4 ± 0.7 3.6 ± 0.2 hy- 28 0.4 ± 0.0 0.8 ± 0.4 2.4 ± 0.6 1.2 ±0.1 droxy- buty- rate (mM) Glucose −18 214 ± 24  250 ± 6  610 ± 39  601± 26  (mg/dL) 28 238 ± 16  152 ± 5  696 ± 66  606 ± 59 

Example 11 Generation of a Bi-Specific Antibody

Various bi-specific antibodies are generated for use in practicing themethods of the invention. For example, GCGR-specific antibodies aregenerated in a bi-specific format (a “bi-specific”) in which variableregions binding to distinct ectodomain and/or EC loop epitopes on GCGRare linked together to confer dual-loop specificity within a singlebinding molecule. Appropriately designed bi-specifics may enhanceoverall glucagon receptor blocking efficacy through increasing both GCGRspecificity and binding avidity. Variable regions with specificity forindividual ectodomain epitopes (e.g., segments of the N-terminal domain,or of the EC1, EC2, or EC3 GCGR loops) or that can bind to differentregions within one ectodomain segment or loop are paired on a structuralscaffold that allows each variable region to bind simultaneously to theseparate epitopes, or to different regions within one ectodomain or ECloop. In one example for a bi-specific, heavy chain variable regions(V_(H)) from a binder with one ectodomain or loop specificity arerecombined with light chain variable regions (V_(L)) from a series ofbinders with a second ectodomain or EC loop specificity to identifynon-cognate V_(L) partners that can be paired with an original V_(H)without disrupting the original specificity for that V_(H). In this way,a single V_(L) segment (e.g., V_(L)1) can be combined with two differentV_(H) domains (e.g., V_(H)1 and V_(H)2) to generate a bi-specificcomprised of two binding “arms” (V_(H)1-V_(L)1 and V_(H)2-V_(L)1). Useof a single V_(L) segment reduces the complexity of the system andthereby simplifies and increases efficiency in cloning, expression, andpurification processes used to generate the bi-specific (See, forexample, U.S. Ser. No. 13/022,759 and US2010/0331527).

Alternatively, antibodies that bind both GCGR and a second target, suchas, but not limited to, for example, human proprotein convertasesubtilisin/kexin type 9 (hPCSK9), may be prepared in a bi-specificformat using techniques described herein, or other techniques known tothose skilled in the art. Antibody variable regions binding to distinctGCGR regions that are extracellularly exposed are linked together withvariable regions that bind to relevant sites on, for example, PCSK9, toconfer dual-antigen specificity within a single binding molecule.Appropriately designed bi-specifics of this nature serve a dualfunction. For example, in the case of a bi-specific antibody that bindsboth GCGR and PCSK9, one may be able to lower blood glucose by virtue ofone arm of the bi-specific antibody binding GCGR, while at the same timelowering plasma lipids, by virtue of the second arm of the antibodybinding PCSK9. Variable regions with specificity for individualectodomain epitopes of GCGR, are combined with a variable region withspecificity for PCSK9 and are paired on a structural scaffold thatallows each variable region to bind to the separate antigens.

The bi-specific binders are tested for binding and functional blockingof the target antigens, for example, GCGR and/or PCSK9, in any of theassays described above for antibodies. For example, standard methods tomeasure soluble protein binding are used to assess the bispecific-PCSK9interaction, such as Biacore, ELISA, size exclusion chromatography,multi-angle laser light scattering, direct scanning calorimetry, andother methods. Binding of bi-specific antibodies to cells expressingGCGR is determined through flow cytometry using a fluorescently labeledsecondary antibody recognizing either or both of the two target antigensbound to cells. Cross-reactivity to the different GCGR ectodomains orloops within and between different species variants is assessed using anELISA binding assay in which synthetic peptides representing thedifferent ectodomains or loops are coated onto the wells of microtiterplates, and binding of a bi-specific is determined through use of asecondary detection antibody. Binding experiments with loop peptides canalso be conducted using surface plasmon resonance experiments, in whichreal-time binding interaction of peptide to antibody is measured byflowing a peptide or bi-specific across a sensor surface on whichbi-specific or peptide, respectively, is captured. Functional in vitroblocking of the GCGR receptor by a bi-specific is determined using anybioassay such as that described herein, or by in vivo determination ofblood glucose levels in appropriate animal models, such as thosedescribed herein. Functional in vitro blocking of PCSK9 by a bi-specificis determined using any bioassay such as that described inWO2010/077854, or in US2010/0166768, or by in vivo determination ofplasma lipid levels in appropriate animal models, such as thosedescribed herein.

Example 12 The Effect of an Anti-GCGR Antibody in aKetamine/Xylazine-Induced Mouse Model of Short Term Stress Hyperglycemia

The effects of H4H1327P anti-GCGR antibody on blood glucose levels weredetermined in an anesthesia (ketamine/xylazine)-induced mouse model ofstress hyperglycemia. The combination of ketamine and xylazine is acommonly used anesthesia for many species including rodents. Ketamine isa dissociative anesthetic, and xylazine is a powerfulsedative/analgesic. Ketamine/xylazine anesthesia has been shown toinduce stress hormone release and elevate blood glucose levelstemporarily (Saha J K et al., Experimental Biology and Medicine 2005,230:777-784). Baseline blood glucose levels were measured in seven2-month-old male C57BL6 mice, and the mice received intramuscularinjection of a combination of ketamine (120 mg/kg) and xylazine (5mg/kg). All mice became unconscious within 5 minutes ofketamine/xylazine injection. Based on the blood glucose levels measuredearlier, mice were divided into 2 groups of n=3 or n=4. Thirty minutesafter ketamine/xylazine administration, while mice were stillunconscious, each group received intravenous injections of the anti-GCGRantibody designated H4H1327P (n=4) or the hlgG isotype control antibodydesignated H1H316P (n=3) at 10 mg/kg. 45-60 minutes afterketamine/xylazine administration, all mice regained consciousness. Micewere bled retroorbitally 0.5, 1.5, 3, 4.5, 6 and 24 hours afterketamine/xylazine administration (=immediately before and 1, 2.5, 4,5.5, and 23.5 hours after mAb injections) for blood glucosemeasurements. Mean±SEM of blood glucose levels at each time point arecalculated for each group and shown in Table 14.

Tabulated Data Summary:

TABLE 14 Time Isotype Anti-GCGR Ab (hrs) control H4H1327P Blood 0 197 ±6  200 ± 6  Glucose 0.5 247 ± 27 244 ± 10 (mg/dL) 1.5 432 ± 23 184 ± 6 3 502 ± 18 267 ± 19 4.5 478 ± 36 273 ± 53 6 330 ± 54 230 ± 52 24 172 ±2  118 ± 8 

Results Summary and Conclusions:

A ketamine/xylazine-induced increase in blood glucose levels wasobserved in mice treated with control mAb between 1.5 and 6 hours afterketamine/xylazine administration. In contrast, mice treated with theanti-GCGR antibody, H4H1327P, showed minimum changes in blood glucoselevels. These data suggest that H4H1327P, within 1 hour ofadministration, blocks ketamine/xylazine-induced hyperglycemia. Theresults are also depicted in FIG. 7.

Example 13 The Effects an Anti-GCGR Antibody in Combination with Humulinon Blood Glucose in Ketamine/Xylazine-Treated Mice

Using the stress hyperglycemia animal model described above, a study wasconducted to determine the effect of therapy with insulin and theanti-GCGR antibody H4H1327P alone, or combined together, on bloodglucose levels.

In this study a total of 23 mice were divided into 6 groups. All micereceived an IM injection of a combination of ketamine (120 mg/kg) andxylazine (5 mg/kg). All mice became unconscious within 5 minutes ofketamine/xylazine injection and regained consciousness 45-60 minutesafter ketamine/xylazine administration. Thirty minutes afterketamine/xylazine administration, Groups 1 (n=3), 2 (n=4), 3 (n=4), and4 (n=4) received an IV injection (via the tail) of the hlgG isotype(negative control) antibody, REGN496, (n=3) at 5 mg/kg, and Groups 5(n=4) and 6 (n=4) received an IV injection (via the tail) of ant-GCGRantibody, H4H1327P at 5 mg/kg. One hour after ketamine/xylazineadministration, Groups 2, 3, 4, and 6 received an SC injection ofHumulin R (Lilly) at 0.1, 0.033, 0.01, and 0.01 U/kg, respectively, at avolume of 5 μL per gram of body weight. Groups 1 and 5 received an SCinjection of water at 5 μL per gram of body weight. Water was used todilute the insulin and serves as a vehicle control. Nonfasted bloodglucose was measured using an ACCU-CHEK® Compact Plus glucometer (Roche)at 0, 1.5, 3, 4.5, 6, 7.5 and 24 h post-ketamine/xylazineadministration. Mice were sorted into antibody treatment groups based ontheir baseline glucose reading so that the mean glucose level acrosstreatment groups was approximately equal. The 6 treatment groups aresummarized in Table 15.

TABLE 15 Treatment group details for H4H1327P and Humulin combinationstudy Treatments Antibody treatment IV Humulin SC (5 mg/kg) (U/kg)Treatment Anesthesia (30 minutes (60 minutes group # (Time 0)post-anesthesia) post-anesthesia) 1 Ketamine/xylazine REGN496 (control)Water 2 Ketamine/xylazine REGN496 (control) 0.1 3 Ketamine/xylazineREGN496 (control) 0.033 4 Ketamine/xylazine REGN496 (control) 0.01 5Ketamine/xylazine H4H1327P Water 6 Ketamine/xylazine H4H1327P 0.01

Data Analyses

For blood glucose data, treatment group values are plotted asmean±standard error of the mean (SEM). Statistical analyses wereperformed utilizing Graph Pad software Prism 5.0 (Macintosh Version).Blood glucose data were initially analyzed by two-way analysis ofvariance (ANOVA); a threshold of p<0.05 was considered statisticallysignificant. After a significant F ratio was obtained with two-wayANOVA, post hoc analysis was conducted between groups with Bonferronipost-tests.

Results

Baseline glucose levels in mice before ketamine/xylazine injection wereapproximately 140 mg/dL (FIG. 8, 0 hr). In mice treated with controlantibody and water, blood glucose levels increased more than 2-fold at90 minutes post-ketamine/xylazine injection and then steadily declined,returning to baseline levels by 6 h post-ketamine/xylazine injection. Incontrast, mice treated with 5 mg/kg H4H1327P and water (H4H1327Pmonotherapy) showed no hyperglycemic response to ketamine/xylazineinjection and blood glucose levels remained stable throughout the study(See FIG. 8).

Mice treated with control antibody and insulin, showed a dose-dependentreduction in blood glucose levels compared with mice treated withcontrol antibody and water. The lowest dose of insulin (0.01 U/kg)caused a trend toward reduced blood glucose levels at 90 minutes and 3hr post-ketamine/xylazine injection, but this was not statisticallysignificant compared with mice treated with control antibody and water.The 2 higher insulin dose groups (0.033 and 0.1 U/kg) caused significantreductions in blood glucose at 90 minutes, 3 hr and 4.5 hr (0.1 U/kgonly) post-ketamine/xylazine injection compared with mice treated withcontrol antibody and water. However, both 0.033 and 0.1 U/kg doses ofinsulin caused blood glucose levels to fall below baseline glucoselevels at 3 h post-ketamine/xylazine injection.

The anti-GCGR antibody H4H1327P was tested in combination with thelowest dose of insulin (0.01 U/kg), which by itself did notsignificantly affect blood glucose levels. The combination of 5 mg/kgH4H1327P and 0.01 U/kg insulin significantly decreased blood glucoselevels at 90 minutes, 3 hr and 4.5 hr post-ketamine/xylazine injection,compared with mice treated with control antibody and water. However, incontrast to H4H1327P monotherapy, blood glucose levels fell belowbaseline levels at 3 hr and 4.5 hr.

Summary

In agreement with the findings of Saha et al., (Saha et al., (2005),Experimental Biology and Medicine 230 (10):777-84) in rats, theanesthetic combination of ketamine/xylazine induced a markedhyperglycemic response in mice that lasted for up to 6 hours. A singledose of 5 or 10 mg/kg of the anti-GCGR antibody H4H1327P prevented thehyperglycemic response to ketamine/xylazine anesthesia with a rapidonset of action; a significant reduction in blood glucose was observedwithin 1 hour and was sustained for up to 4 hours post-H4H1327Padministration. Further, H4H1327P monotherapy effectively reduced andstabilized blood glucose in this model without causing glucose to fallbelow baseline values. In contrast, insulin monotherapy (at 0.033 U/kgor 0.1 U/kg) or H4H1327P and insulin combination therapy were both ableto reduce blood glucose but caused glucose levels to fall below baselinevalues.

To summarize, the study presented here demonstrated a beneficial effectof combined treatment of insulin plus the anti-GCGR antibody of theinvention, H4H1327P. In contrast to treatment with insulin alone (whichmay result in a possible hypoglycemic response, which could result in anincrease in morbidity or mortality), the use of the anti-GCGR antibody,H4H1327P in conjunction with insulin has an insulin sparing effect inthis animal model of stress hyperglycemia. Based on these findings, itmay be possible to lower the dose of insulin needed (or shorten theduration of use) if therapy is combined with the anti-GCGR antibody ofthe invention, H4H1327P. Furthermore, treatment with H4H1327Pmonotherapy prevents ketamine/xylazine-induced hyperglycemia withoutcausing hypoglycemia. Accordingly, these findings support the use ofH4H1327P as stand alone therapy, or for use as adjunct therapy withinsulin in perioperative stress hyperglycemia.

Example 14 Single Dosing of H4H1327P in Cynomolgus Monkeys withSpontaneous Diabetes

A study was done to determine the effect of a single dose of mAbH4H1327P, at two dose levels, on glucose under fasting, fed, and oraland IV glucose challenge conditions in cynomolgus monkeys withspontaneous diabetes. In addition, a second objective of this study wasto estimate the onset and duration of a glucose lowering effect in orderto assess the feasibility of pursuing the use of the antibodies forshort-term indications, where glucose lowering in the first hours afteradministration is desired. Safety endpoints (lipids, liver enzymes, andpancreatic enzymes) and PK were also evaluated.

Twenty-six cynomolgus monkeys (12 males and 14 females, ages between 11and 21-years-old, body weights between 4.7 and 14.9 kg) with a historyof diabetes and/or pre-diabetes were first screened for the presence ofmild fasting hyperglycemia (blood glucose >90 mg/dL), and 15 animalswere selected. Next, a 60 minute intravenous glucose tolerance test(ivGTT) (0.25 g glucose/kg body weight) was performed following anovernight fasting to select animals with glucose intolerance (glucoseAUC >8500 mg/dL×min). Blood samples were collected 5, 10, 20, 40, and 60minutes after glucose administration, and all procedures were performedunder ketamine anesthesia. Ten out of the 15 hyperglycemic animalssatisfied the glucose intolerance criteria and these monkeys were usedfor the study described below and also depicted in FIG. 9.

Methods and Results

5 days prior to H4H1327P administration, an oral glucose tolerance test(oGTT) (1.75 g glucose/kg body weight) was performed following anovernight fasting to establish baseline oGTT data. Blood samples werecollected 15, 30, 60, 90, 120, and 180 min after glucose administrationfor blood glucose measurements, and no anesthesia was used throughoutthe procedure. 2 days prior to H4H1327P administration, 30 and 60minutes post-meal blood glucose levels following an overnight fastingwere measured to establish baseline post-meal blood glucose values. Noanesthesia was used throughout the procedure.

The 10 selected monkeys were divided into two groups of 5 animals eachbased on the baseline fasting blood glucose levels and ivGTT and oGTTglucose AUC data. The monkeys were fasted overnight, and fasting bloodwas collected for serum chemistry immediately prior to H4H1327Padministration. The animals received an intravenous bolus administrationof H4H1327P at 20 mg/kg (n=5) or 5 mg/kg (n=5). Immediately afterH4H1327P dosing, ivGTT was performed to determine the onset of H4H1327Paction. Blood samples were collected 5, 10, 20, 40, and 60 min afterglucose administration for blood glucose and plasma insulin, andglucagon measurements. All procedures were performed under ketamineanesthesia.

Two animals treated with 20 mg/kg H4H1327P and one animal treated with 5mg/kg H4H1327P were excluded from the study analysis as they exhibitedTime 0 (fasting) blood glucose levels equal to or below 90 mg/dL. SinceTime 0 glucose levels of ivGTT immediately after H4H1327P administrationwere measured prior to H4H1327P dosing, and there was slight reductionin group mean Time 0 glucose levels in comparison to the levels duringbaseline ivGTT, % of Time 0 glucose level was calculated for glucoselevel of each time point for each animal. Using the % glucose, areaunder the curve (AUC) from the beginning to the end of ivGTT andfractional (20 or 40 min interval) glucose AUC were calculated for eachanimal. Mean±SEM of % glucose AUC and fractional % glucose AUC werecalculated for each group. Mean % glucose AUC of the entire 60 min andmean fractional % glucose AUC of 20-60 and 40-60 min intervals weresignificantly reduced in monkeys treated with 5 mg/kg H4H1327P (Table17). Mean fractional % glucose AUC of 40-60 min interval wassignificantly reduced in monkeys treated with 20 mg/kg H4H1327P (Table16). Mean±SEM of plasma insulin and glucagon levels were calculated foreach group for each blood collection time points. No significant changesin mean plasma insulin or glucagon levels were observed during theivGTT, in comparison to the levels during baseline ivGTT, in bothtreatment groups. Statistical analysis was performed with Prism software(version 5). To assess the significance before and after H4H1327Padministration, paired T-test was used. *: p<0.05, **: p<0.01, ***:p<0.001, ****: p<0.000.

Two days after H4H1327P administration, 30 and 60 minutes post-mealblood glucose levels following an overnight fast were measured. Mean±SEMof 30 and 60 minutes post-meal glucose levels were calculated for eachgroup. Significant reduction in mean 60 min post-meal blood glucoselevel was detected in monkeys treated with 20 mg/kg H4H1327P (Table 18).Although the data did not reach statistical significance, trends towardreduction in 30 min post-meal blood glucose levels in monkeys treatedwith 20 mg/kg H4H1327P (Table 18) and 30 and 60 min post-meal bloodglucose levels in monkeys treated with 5 mg/kg H4H1327P were observed(Table 19). It is important to note that the greatest post-meal glucosereductions were seen in animals with the highest glucose at baseline.The lowest post-meal blood glucose recorded was 86 mg/dL in an animaltreated with 20 mg/kg H4H1327P. Statistical analysis was performed withPrism software (version 5). To assess the significance before and afterH4H1327P administration, paired T-test was used. *: p<0.05, **: p<0.01,***: p<0.001, ****: p<0.0001.

Four days after H4H1327P administration, oGTT was performed in consciousanimals following an overnight fasting. Blood samples were collected 15,30, 60, 90, 120, and 180 min after glucose administration for bloodglucose measurements. Mean±SEM of fasting blood glucose levels werecalculated for each group. Significant (p=0.0045) and near-significant(p=0.052) reductions in mean fasting blood glucose levels were observedin monkeys treated with 20 and 5 mg/kg H4H1327P, respectively (Table20). The lowest fasting blood glucose recorded was 67 mg/dL in an animaltreated with 20 mg/kg H4H1327P. Mean±SEM of blood glucose levels at eachtime point during oGTT were calculated for each group. Blood glucose AUCduring oGTT was calculated for each animal, and mean±SEM of bloodglucose AUC were calculated for each group. A significant reduction inmean glucose AUC was observed in monkeys treated with 20 mg/kg H4H1327P.Trends toward reductions in glucose AUC were observed in monkeys treatedwith 5 mg/kg H4H1327P (Table 21). Statistical analysis was performedwith Prism software (version 5). To assess the significance before andafter H4H1327P administration, paired T-test was used. *: p<0.05, **:p<0.01, ***: p<0.001, ****: p<0.0001.

Seven days after H4H1327P administration, ivGTT was performed inketamine anesthetized animals following an overnight fast. Blood sampleswere collected 5, 10, 20, 40, and 60 min after glucose administrationfor blood glucose and plasma insulin and glucagon measurements. Mean±SEMof fasting blood glucose levels were calculated for each group. Asignificant reduction in mean fasting blood glucose level was observedin monkeys treated with 20 mg/kg H4H1327P (Table 22). The lowest fastingblood glucose recorded was 79 mg/dL in an animal treated with 20 mg/kgH4H1327P. Mean±SEM of blood glucose levels at each time point duringivGTT were calculated for each group. Blood glucose AUC during ivGTT wascalculated for each animal, and mean±SEM of blood glucose AUC werecalculated for each group.

Seven days post H4H1327P administration, a significant reduction in meanglucose AUC was observed in monkeys treated with 20 mg/kg H4H1327P(Table 23). Non-significant reductions in glucose AUC were also observedin monkeys treated with 5 mg/kg H4H1327P (Table 23). Mean±SEM of plasmainsulin and glucagon levels at each blood sampling time point werecalculated for each group. Although they did not reach statisticalsignificance, in both treatment groups, circulating insulin levelsappear to be elevated after glucose administration 7 days post H4H1327Padministration in comparison to the levels during baseline ivGTT. Inboth treatment groups, mean glucagon levels were significantly increasedbefore and after glucose administration 7 days post H4H1327Padministration in comparison to baseline. Immediately prior to glucoseadministration, fasting blood was collected for serum chemistry.Mean±SEM of each serum parameter were calculated for each group.Significant increases in serum triglyceride levels were observed inmonkeys treated with 20 mg/kg H4H1327P 7 days post H4H1327Padministration. Trends toward an increase in serum triglyceride levelswere also observed in monkeys treated with 5 mg/kg H4H1327P 7 days postH4H1327P administration in comparison to baseline.

Statistical analysis was performed with Prism software (version 5). Toassess the significance before and after H4H1327P administration, pairedT-test was used. *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001.

TABLE 16 Fractional % Glucose AUC (%*min), 20 mg/kg (n = 3) Baseline Day0 Mean SEM Mean SEM  0-20 min 2752 57 2715 205 20-40 min 2500 72 2388149 40-60 min 2240 71 2009 103

TABLE 17 Fractional % Glucose AUC (%*min), 5 mg/kg (n = 4) Baseline Day0 Mean SEM Mean SEM  0-20 min 2814 249 2797 275 20-40 min 2467 100 2329137 40-60 min 2199 62 1898 72

TABLE 18 30 and 60 min Post-meal Glucose (mg/dL), 20 mg/kg (n = 5)Baseline Day 2 Mean SEM Mean SEM 30 min 208 44 112 12 60 min 235 44 13112

TABLE 19 30 and 60 min Post-meal Glucose (mg/dL), 5 mg/kg (n = 4)Baseline Day 2 Mean SEM Mean SEM 30 min 240 41 166 23 60 min 287 53 19930

TABLE 20 Fasting Glucose Before oGTT (mg/dL) Baseline Day 4 Mean SEMMean SEM 20 mg/kg 153 16 83 9 REGN1193  5 mg/kg 178 35 131 22 REGN1193

TABLE 21 oGTT Glucose AUC (mg/dL*min) Baseline Day 4 Mean SEM Mean SEM20 mg/kg 38181 6302 24597 3461 REGN1193  5 mg/kg 40656 7656 34044 5056REGN1193

TABLE 22 Fasting Glucose Before ivGTT (mg/dL) Baseline Day 7 Mean SEMMean SEM 20 mg/kg 167 22 86 4 REGN1193  5 mg/kg 176 28 150 27 REGN1193

TABLE 23 ivGTT Glucose AUC (mg/dL*min) Baseline Day 7 Mean SEM Mean SEM20 mg/kg 12633 1481 8112 385 REGN1193  5 mg/kg 13472 1871 12100 1613REGN1193Summary of In Vivo Pharmacology Data in the Cynomolgus Monkey Study withH4H1327P

The objective of this study was to evaluate the effects of H4H1327P onblood glucose levels, glucose tolerance, and other serum parameters,when administered at two different doses in cynomolgus monkeys withspontaneous diabetes.

In animals treated with 20 mg/kg H4H1327P, 46% (p=0.04) and 50% (p=0.01)reductions in fasting blood glucose levels were observed 4 and 7 dayspost H4H1327P administration, respectively, in comparison to baselinevalues. Post-meal blood glucose levels measured 2 days post H4H1327Padministration were 46% (p=0.03) reduced. During an ivGTT performedimmediately after H4H1327P administration, % glucose AUC of 40-60 mininterval was reduced by 10% (p=0.02), suggesting the onset of H4H1327Paction on blood glucose is ˜1 h. At oGTT and ivGTT performed 4 and 7days after H4H1327P administration, 35% (p=0.01) and 36% (p=0.02)reductions in glucose AUC were observed, respectively.

In animals treated with 5 mg/kg H4H1327P, 26% (p=0.052) and 15% (p=0.2)reductions in fasting blood glucose levels were observed 4 and 7 dayspost H4H1327P administration, respectively, in comparison to baselinevalues. Post-meal blood glucose levels measured 2 days post H4H1327Padministration were 30% (p=0.08) reduced. During an ivGTT performedimmediately after H4H1327P administration, % glucose AUC of 40-60 mininterval was reduced by 14% (p=0.01). At oGTT and ivGTT performed 4 and7 days after H4H1327P administration, non-significant 20% (p=0.2) and10% (p=0.3) reductions in glucose AUC were observed, respectively. Inboth dose groups, there were no changes in plasma insulin or glucagonconcentrations during ivGTT performed before and immediately afterH4H1327P administration. However, trends toward increases in plasmainsulin levels after glucose administration were observed during theivGTT performed 7 days post H4H1327P administration, suggesting elevatedglucose induced insulin secretion in H4H1327P treated animals. 7 dayspost H4H1327P administration, plasma glucagon levels were increased by5.5- (p<0.001) and 4-fold (p<0.001) with 20 and 5 mg/kg H4H1327P,respectively. 1.8- (p=0.043) and 4.3-fold (p=0.119) increases in serumtriglyceride levels were also observed in monkeys treated with 20 and 5mg/kg H4H1327P. It is of note that in no case did fasting blood glucoselevels of H4H1327P treated animals fall below normal monkey range (50-70mg/dL).

In conclusion, the anti-GCGR mAb H4H1327P reduced blood glucose levelsand improved glucose tolerance in monkeys with spontaneous diabetes in adose-dependent manner, demonstrating that H4H1327P has glucose-loweringefficacy in non-human primates. H4H1327P onset of action was observedwithin 1 hour after IV bolus administration of the mAb, supporting theuse of H4H1327P for short-term indications. H4H1327P also increasedglucose induced insulin secretion without changes in LDL cholesterollevels 7 days after drug administration. No differences in body weightwere detected in all animals after H4H1327P administration andthroughout the study. These data in diabetic monkeys support theclinical use of H4H1327P as a glucose lowering agent for both long andshort term indications.

Example 15 A Proposed Clinical Study to Determine the Effect of anAnti-GCGR Antibody on Stress Hyperglycemia in Human Patients

Two distinct programs are proposed for testing the effects of ananti-GCGR antibody in patients with stress hyperglycemia. These programsare as follows:

Study Plan A)

a development program for H4H1327P in stress hyperglycemia for criticalpatients in the ICU using composite metabolic endpoints as a surrogateendpoint and a post approval morbidity and mortality outcome study.

Study Plan B)

a clinical development program in patients with stress hyperglycemia innon-critical ICU patients with metabolic endpoints, insulin dose, andresource utilization as key endpoints. Morbidity and mortality will bedesignated as other secondary endpoints since this population is at alow risk for death.

Position

According to 2012 guidelines, stress hyperglycemia in the diabetic andnon-diabetic hospitalized patient is considered a medical conditionrequiring prompt therapeutic intervention (Korytkowski M, McDonnell M E,Umpierrez G E, Zonszein J, J Clin Endocrinol Metab. (2012), January;97(1):27A-8A; Umpierrez G E, Hellman R, Korytkowski M T, Kosiborod M,Maynard G A, Montori V M, et al. J Clin Endocrinol Metab. (2012)January; 97(1):16-38). Hyperglucagonemia plays a key role in stresshyperglycemia by increasing hepatic glucose output and thuscounterbalancing the glucose lowering effect of insulin. In ICU andnon-ICU patients, elevated blood glucose levels are associated withincreased morbidity and mortality (Baker E H, Janaway C H, Philips B J,Brennan A L, Baines D L, Wood D M, et al., Thorax, (2006), April;61(4):284-9), Umpierrez G E, Isaacs S D, Bazargan N, You X, Thaler L M,Kitabchi A E, J Clin Endocrinol Metab. (2002), March 87(3):978-82;McAlister F A, Majumdar S R, Blitz S, Rowe B H, Romney J, Marrie T J,Diabetes Care. (2005), April 28(4):810-5). Eighty percent of ICUpatients with stress hyperglycemia have no history of diabetes beforehospital admission. Patients with new hyperglycemia (induced ordiagnosed at the time of hospitalization) have significantly higherin-hospital mortality rate and worse functional outcome than patientswith a prior history of diabetes and subjects with normoglycemia.Current treatment for stress hyperglycemia is insulin, which is quiteeffective in controlling glucose but tight glucose control is oftenassociated with episodes of hypoglycemia. Therefore, an unmet medicalneed exists for effective treatment with no associated hypoglycemiairrespective of the underlying critical illness. We hypothesize that forthe treatment of stress hyperglycemia, H4H1327P in combination withinsulin compared to insulin alone will be effective in decreasingmorbidity and mortality through improved metabolic control, anindication that addresses an unmet medical need.

A Phase 2 dose ranging study is planned in ICU patients (randomized 1:1to H4H1327P or placebo) to assess the efficacy and safety of H4H1327P.For the Phase 3, a study is planned whereby-patients will be randomized1:1 to H4H1327P (treatment arms will be added depending on the number ofdoses) or placebo using a surrogate primary endpoint that would mostlikely predict morbidity and mortality benefit. Mackenzie et. al.demonstrated that the combination of metrics of central tendency(average glucose), variability of glucose (excursions) and hypoglycemia(minimum glucose) are better predictors of mortality than the individualmetrics alone (Table 24), (MacKenzie, IM et al., Intensive Care Med.(2011), March, 37(3):435-43) In addition, (1) the interaction betweenglucose concentrations and outcome may arise from these threeindependent and synergistic domains, namely, central tendency,variability, and minimum value; (2) the relationship may be non-linearand specific to both patient population (medical or surgical) anddomain; (3) the relationship has a dose-response component meaning thelonger the metric is met, the better the outcome.

Thus, the goal for therapy with an anti-GCGR antibody, as describedherein, in combination with insulin, is to maintain a constant level ofglucose control in the target range, devoid of glucose swings, both highand low.

TABLE 24 Mean and variability of glucose and hypoglycemia are betterpredictors of mortality than individual metrics alone Low risk High riskOR (95% CI) Death % Death % Low vs High 1. Average glucose 11.4 22.5 2.0(1.6, 2.5) 2. Glucose excursions 9.9 19.4 2.2 (1.8, 2.7) 3. minimumglucose 9.3 20.5 2.5 (2.0, 3.1) 1 + 2 7.8 23.4 3.6 (2.6, 4.9) 1 + 3 6.224.2 4.8 (3.4, 6.8) 2 + 3 7.6 24.2 3.9 (2.9, 5.2) 1 + 2 + 3 6.0 27.8 6.0(3.9, 9.2)

We propose that the study in critically ill patients with stresshyperglycemia, assuming it meets key endpoints, would be followed by apost-approval morbidity and mortality outcome study in ICU patients.

B. Study Plan B. Study Population Under Consideration

The population under consideration is patients 18-65 years of age withnon-critical illness (not in the ICU) and stress hyperglycemia and whohave a blood glucose>180 mg/dL and expected length of hospital stay thatexceeds 5 days are eligible for the proposed study. Patients will not bein the ICU but may be admitted to monitored (e.g., telemetry or diabetesunit) beds or general ward beds. Patients may or may not have a historyof diabetes mellitus. Patients admitted for DKA and HHS are not eligiblefor this study.

Study Design Under Consideration

The design under consideration is a randomized double blind placebocontrolled parallel study in patients with non-critical stresshyperglycemia. Subjects will be randomized to H4H1327P or placebo, inaddition to their usual care. Based on the current practice guidelines,providers are expected to treat blood glucoses of 140-180 mg/dL withbasal/bolus insulin. They are also expected to reassess patients andtreatment plans when blood glucose <100 mg/dL and to alter treatmentwhen blood glucose <70 mg/dL. In this trial, patients will be randomizedif their blood glucose is >180 mg/dL to receive insulin (basal/bolus)and H4H1327P or insulin (basal/bolus) and placebo for H4H1327P (numberof arms and doses to be determined) in a 1:1 ratio). The insulin willnot be blinded. Patients will be followed until discharge. The primaryendpoints of metabolic control and insulin dose will be collected for 72hr after randomization.

What is claimed is:
 1. A method of preventing the onset of stresshyperglycemia in a patient, or for treating a patient suffering fromstress hyperglycemia, the method comprising administering to a patient atherapeutically effective amount of a composition comprising a glucagonreceptor antagonist, wherein the patient exhibits elevated levels ofblood glucose caused or exacerbated by one or more stress-inducingstimulus or glucose elevating stimulus.
 2. The method of claim 1,wherein the patient is identified on the basis of having a blood glucoselevel greater than about 140 mg/dL.
 3. The method of claim 1, whereinthe stress-inducing stimulus or glucose elevating stimulus is selectedfrom the group consisting of pre-existing type 1 or type 2 diabetes,hypertonic dehydration, infusion of catecholamine pressors, parenteralnutrition, enteral nutrition, glucocorticoid therapy, obesity, aging,excessive dextrose administration, pancreatitis, sepsis, stroke,traumatic head injury, hypothermia, hypoxemia, uremia, cirrhosis,anesthesia, pre-operative or post-operative hospital stays(peri-operative hyperglycemia), admission to an emergency room, a traumacenter, or an intensive care unit, prolonged hospital stays, surgicalprocedures, an infection and a chronic illness.
 4. The method of claim1, wherein the glucagon receptor antagonist is an isolated humanmonoclonal antibody, or an antigen binding fragment thereof, specificfor the human glucagon receptor, and wherein the isolated humanmonoclonal antibody or the antigen-binding fragment thereof is capableof blocking the binding of glucagon to the glucagon receptor.
 5. Themethod of claim 4, wherein the isolated human monoclonal antibody orantigen-binding fragment thereof comprises the complementaritydetermining regions (CDRs) of a heavy chain variable region (HCVR),wherein the HCVR has an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126,130 and 146; and the CDRs of a light chain variable region (LCVR),wherein the LCVR has an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128,138 and
 148. 6. The method of claim 5, wherein the isolated humanmonoclonal antibody or antigen-binding fragment thereof comprises aheavy chain variable region (HCVR) having an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 70,86, 90, 106, 110, 126, 130 and
 146. 7. The method of claim 5, whereinthe isolated human monoclonal antibody or antigen-binding fragmentthereof comprises a light chain variable region (LCVR) having an aminoacid sequence selected from the group consisting of SEQ ID NO: 10, 26,42, 58, 68, 78, 88, 98, 108, 118, 128, 138 and
 148. 8. The method ofclaim 5, wherein the isolated human monoclonal antibody orantigen-binding fragment thereof comprises: (a) a HCVR having an aminoacid sequence selected from the group consisting of SEQ ID NO: 2, 18,34, 50, 66, 70, 86, 90, 106, 110, 126, 130 and 146; and (b) a LCVRhaving an amino acid sequence selected from the group consisting of SEQID NO: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138 and
 148. 9.The method of claim 5, wherein the isolated human monoclonal antibody orantigen-binding fragment thereof comprises: (a) a HCDR1 domain having anamino acid sequence selected from the group consisting of SEQ ID NO: 4,20, 36, 52, 72, 92, 112 and 132; (b) a HCDR2 domain having an amino acidsequence selected from the group consisting of SEQ ID NO: 6, 22, 38, 54,74, 94, 114 and 134; (c) a HCDR3 domain having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 76, 96,116 and 136; (d) a LCDR1 domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 12, 28, 44, 60, 80, 100, 120 and140; (f) a LCDR2 domain having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 14, 30, 46, 62, 82, 102, 122 and 142; and(g) a LCDR3 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 16, 32, 48, 64, 84, 104, 124 and
 144. 10. Themethod of claim 5, wherein the isolated human monoclonal antibody orantigen-binding fragment comprises a HCVR/LCVR sequence pair selectedfrom the group consisting of SEQ ID NO: 2/10, 18/26, 34/42, 50/58,66/68, 70/78, 86/88, 90/98, 106/108, 110/118, 126/128, 130/138, and146/148.
 11. The method of claim 1, wherein the patient is a diabeticpatient.
 12. The method of claim 11, wherein the diabetic patient is acritically ill diabetic patient.
 13. The method of claim 11, wherein thediabetic patient is a non-critically ill diabetic patient.
 14. Themethod of claim 11, wherein the diabetic patient is administered one ormore additional therapeutic agents selected from the group consisting ofinsulin, a biguanide (metformin), a sulfonylurea (such as glyburide,glipizide), a PPAR gamma agonist (pioglitazone, rosiglitazone), an alphaglucosidase inhibitor (acarbose, voglibose), SYMLIN® (pramlintide), anyGLP-1 compound, or an analog, an agonist, a derivative, or asecretagogue thereof, and a dipeptidyl peptidase IV inhibitor.
 15. Themethod of claim 1, wherein the patient is a non-diabetic patient. 16.The method of claim 15, wherein the non-diabetic patient is a criticallyill non-diabetic patient.
 17. The method of claim 15, wherein thenon-diabetic patient is a non-critically ill non-diabetic patient. 18.The method of claim 15, wherein the non-diabetic patient is administeredinsulin as a second therapeutic agent.
 19. The method of claim 4,wherein the administering of the antibody results in lowering the bloodglucose level to between about 80 mg/dL to about 140 mg/dL.
 20. Themethod of claim 19, wherein the administering of the antibody results inlowering the blood glucose level to between about 80 mg/dL to about 110mg/dL.
 21. The method of claim 19, wherein the administering of theantibody results in lowering the blood glucose level to between about100 mg/dL to about 140 mg/dL.
 22. The method of claim 1, wherein theadministering results in lowering blood glucose levels without the riskof inducing hypoglycemia.
 23. The method of claim 1, wherein theadministering results in a decreased risk for infection, organ failure,disability from stroke, arrhythmia, or death.
 24. A method of reducingthe amount/dosage of insulin necessary to lower blood glucose levels toa normal range in a patient at risk for developing stress hyperglycemia,or in a patient suffering from stress hyperglycemia, the methodcomprising administering an isolated human monoclonal antibody thatbinds specifically to the glucagon receptor concomitantly with insulin.25. The method of claim 24, wherein the amount/dosage of insulin may bereduced by about 30% to about 95% when administered concomitantly withan isolated human monoclonal antibody that binds specifically to theglucagon receptor.
 26. The method of claim 25, wherein the amount/dosageof insulin may be reduced by about 90% when administered concomitantlywith an isolated human monoclonal antibody that binds specifically tothe glucagon receptor.
 27. The method of claim 24, wherein the isolatedhuman monoclonal antibody comprises the complementarity determiningregions (CDRs) of a heavy chain variable region (HCVR), wherein the HCVRhas an amino acid sequence selected from the group consisting of SEQ IDNOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130 and 146; and theCDRs of a light chain variable region (LCVR), wherein the LCVR has anamino acid sequence selected from the group consisting of SEQ ID NOs:10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138 and
 148. 28. Themethod of claim 27, wherein the isolated human monoclonal antibody orantigen-binding fragment thereof comprises a heavy chain variable region(HCVR) having an amino acid sequence selected from the group consistingof SEQ ID NO: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130 and 146.29. The method of claim 27, wherein the isolated human monoclonalantibody or antigen-binding fragment thereof comprises a light chainvariable region (LCVR) having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118,128, 138 and
 148. 30. The method of claim 27, wherein the isolated humanmonoclonal antibody or antigen-binding fragment thereof comprises: (a) aHCVR having an amino acid sequence selected from the group consisting ofSEQ ID NO: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130 and 146;and (b) a LCVR having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128,138 and
 148. 31. The method of claim 27, wherein the isolated humanmonoclonal antibody or antigen-binding fragment thereof comprises: (a) aHCDR1 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, 20, 36, 52, 72, 92, 112 and 132; (b) a HCDR2domain having an amino acid sequence selected from the group consistingof SEQ ID NO: 6, 22, 38, 54, 74, 94, 114 and 134; (c) a HCDR3 domainhaving an amino acid sequence selected from the group consisting of SEQID NOs: 8, 24, 40, 56, 76, 96, 116 and 136; (d) a LCDR1 domain having anamino acid sequence selected from the group consisting of SEQ ID NO: 12,28, 44, 60, 80, 100, 120 and 140; (e) a LCDR2 domain having an aminoacid sequence selected from the group consisting of SEQ ID NO: 14, 30,46, 62, 82, 102, 122 and 142; and (f) a LCDR3 domain having an aminoacid sequence selected from the group consisting of SEQ ID NO: 16, 32,48, 64, 84, 104, 124 and
 144. 32. The method of claim 27, wherein theisolated human monoclonal antibody or antigen-binding fragment comprisesa HCVR/LCVR sequence pair selected from the group consisting of SEQ IDNO: 2/10, 18/26, 34/42, 50/58, 66/68, 70/78, 86/88, 90/98, 106/108,110/118, 126/128, 130/138, and 146/148.
 33. The method of claim 27,wherein the isolated human monoclonal antibody comprises a HCVR/LCVRamino acid sequence pair as set forth in SEQ ID NOs: 86/88.